Phytase variants

ABSTRACT

The present invention relates to a phytase which has at least 74% identity to a phytase derived from  Citrobacter braakii  and comprises at least one alteration as compared to this phytase. These phytase variants have amended, preferably improved, properties, such as thermostability, temperature profile, pH profile, specific activity, performance in animal feed, reduced protease sensibility, and/or an amended glycosylation pattern. The invention also relates to DNA encoding these phytases, methods of their production, as well as the use thereof, e.g., in animal feed and animal feed additives.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No. 14/504,517filed on Oct. 2, 2014, now U.S. Pat. No. 9,451,783, which is adivisional of U.S. application Ser. No. 13/874,954 filed on May 1, 2013,now U.S. Pat. No. 8,877,471, which is a divisional of U.S. applicationSer. No. 12/294,526 filed on Nov. 5, 2008, now U.S. Pat. No. 8,460,656,which is a 35 U.S.C. 371 national application of internationalapplication no. PCT/DK2007/000135 filed Mar. 19, 2007, which claimspriority or the benefit under 35 U.S.C. 119 of Danish application nos.PA 2006 00484 and PA 2006 00581 filed Apr. 4, 2006 and Apr. 25, 2006,respectively, and U.S. provisional application Nos. 60/788,966 and60/794,757 filed Apr. 4, 2006 and Apr. 25, 2006, respectively. Thecontent of these applications is fully incorporated herein by reference.

REFERENCE TO SEQUENCE LISTING

This application contains a Sequence Listing in computer readable form.The computer readable form is incorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates to a phytase which has at least 74%identity to a phytase derived from Citrobacter braakii ATCC 51113 andcomprises at least one alteration as compared to this phytase (i.e., isa variant thereof). The invention also relates to DNA encoding thesephytases, methods of their production, as well as the use thereof, e.g.,in animal feed and animal feed additives. The mature part of theCitrobacter braakii ATCC 51113 phytase is included in the sequencelisting as SEQ ID NO: 2.

BACKGROUND OF THE INVENTION Background Art

The sequence of the phyA gene from Citrobacter freundii has beensubmitted by Zinin et al. to the EMBL/GenBank/DDBJ databases withaccession no. AY390262. The corresponding phytase amino acid sequence isfound in the UniProt/TrEMBL databases with accession no. Q676V7. Theexpected mature part of Q676V7 is included in the present sequencelisting as SEQ ID NO: 4.

WO 2004/085638 discloses, as SEQ ID NO: 7, the amino acid sequence of aphytase from Citrobacter braakii YH-15, deposited as KCCM 10427. Themature part of this amino acid sequence is included herein as SEQ ID NO:3. This sequence is also found in the database Geneseqp with accessionno. ADU50737.

WO 2006/037328 discloses the wildtype phytase of Citrobacter braakiiATCC 51113 (i.e., SEQ ID NO: 2 herein), as well as a variant thereof,which is also included in the present sequence listing, viz. as SEQ IDNO: 6.

WO 2006/038062 and WO 2006/038128 both disclose the amino acid sequenceof the phytase gene of Citrobacter freundii P3-42, deposited underaccession number NCIMB 41247. This amino acid sequence is includedherein as SEQ ID NO: 9.

It is an object of the invention to provide phytases of amended,preferably, improved properties. Non-limiting examples of suchproperties are: Thermostability, temperature profile pH profile,specific activity, performance in animal feed, protease-sensibility,and/or glycosylation pattern.

SUMMARY OF THE INVENTION

The present invention relates to a phytase which has at least 74%identity to SEQ ID NO: 2 and which comprises at least one alteration ascompared to SEQ ID NO: 2 in at least one position selected from thefollowing: 1, 2, 3, 4, 5, 31, 41, 46, 52, 53, 55, 57, 59, 74, 76, 82,84, 91, 99, 100, 104, 105, 107, 109, 111, 114, 115, 116, 117, 118, 119,120, 121, 122, 123, 124, 136, 137, 141, 154, 161, 162, 164, 167, 171,176, 177, 179, 180, 181, 182, 183, 184, 185, 186, 196, 199, 200, 202,203, 218, 223, 239, 240, 241, 247, 273, 276, 281, 282, 283, 284, 285,286, 289, 294, 299, 308, 314, 316, 324, 331, 339, 351, 355, 362, 379,385, 406, 409, 410, and 411; with the proviso that the phytase is notSEQ ID NO: 3, not SEQ ID NO: 4, and not SEQ ID NO: 6.

The invention also relates to a phytase which has at least 74% identityto SEQ ID NO: 2 and which comprises at least one of the followingalterations: 1H,K,R, 60P, 105E, 106A,G, 155F, 157F, 173P, 175L, 188P,205P, 215M, 231P, 254Y, 280P, 330D, and/or 371P; with the proviso thatthe phytase is not SEQ ID NO: 3, not SEQ ID NO: 4, not SEQ ID NO: 6, andnot SEQ ID NO: 9 and the variants thereof listed in FIG. 1.

The invention also relates to DNA encoding these phytases, methods oftheir production, as well as the use thereof, e.g., in animal feed andanimal feed additives.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 corresponds to Table 2 of WO 2006/038062 and discloses a numberof variants of the Citrobacter freundii NCIMB 41247 phytase which hasthe amino acid sequence of SEQ ID NO: 9; and

FIG. 2 is an alignment of the phytases of SEQ ID NO: 2 and 9.

The position numbers in FIG. 1 refer to the numbering of SEQ ID NO: 9.The corresponding SEQ ID NO: 2 positions can be found by deduction of 22(e.g., variant P229S of FIG. 1 means variant P207S using the numberingof the present application).

DETAILED DESCRIPTION OF THE INVENTION

In a first aspect, the present invention relates to a phytase which hasat least 74% identity to SEQ ID NO: 2 and which comprises at least onealteration as compared to SEQ ID NO: 2 in at least one position selectedfrom the following: 1, 2, 3, 4, 5, 31, 41, 46, 52, 53, 55, 57, 59, 74,76, 82, 84, 91, 99, 100, 104, 105, 107, 109, 111, 114, 115, 116, 117,118, 119, 120, 121, 122, 123, 124, 136, 137, 141, 154, 161, 162, 164,167, 171, 176, 177, 179, 180, 181, 182, 183, 184, 185, 186, 196, 199,200, 202, 203, 218, 223, 239, 240, 241, 247, 273, 276, 281, 282, 283,284, 285, 286, 289, 294, 299, 308, 314, 316, 324, 331, 339, 351, 355,362, 379, 385, 406, 409, 410, and 411; with the proviso that the phytaseis not SEQ ID NO: 3, not SEQ ID NO: 4, and not SEQ ID NO: 6.

The percentage of identity is determined as described in the section“Phytase Polypeptides, Percentage of Identity”.

The position numbers refer to the position numbering of SEQ ID NO: 2, asdescribed in the section “Position Numbering.” Positions correspondingto these SEQ ID NO: 2 position numbers in other phytases are determinedas described in the section “Identifying Corresponding PositionNumbers.”

The phytase of the invention is a variant of the phytase of SEQ ID NO:2, viz. it is not identical to SEQ ID NO: 2, as it comprises at leastone alteration as compared to SEQ ID NO: 2.

In a particular embodiment, the phytase of the invention comprises atleast one alteration as compared to SEQ ID NO: 2 in at least oneposition selected from the following: 1, 2, 3, 4, 5, 31, 46, 52, 53, 55,57, 59, 76, 82, 99, 100, 107, 109, 111, 114, 115, 116, 117, 118, 119,120, 121, 122, 123, 124, 137, 141, 161, 162, 164, 167, 179, 180, 181,182, 183, 184, 185, 186, 196, 199, 200, 202, 218, 223, 241, 273, 276,285, 286, 299, 314, 331, 339, 362, 379, 385, 406, 410, and 411.

In another particular embodiment the phytase of the invention is not SEQID NO: 9.

In a still further particular embodiment, the phytase of the inventionis not the variants of SEQ ID NO: 9 listed in FIG. 1.

In a preferred embodiment, the phytase of the invention comprises atleast one of the following alterations: 1*, 2*, 3*, 4P, 5P, 31C,T, 41P,46C,D,E, 52C,E, 53V,Q, 55D,I, 57Y, 59C, 74A, 76G, 82E, 84Y, 91C,P, 99C,100C, 104A, 105F, 107D,E,G, 109A,G, 111P, 114H,N,T, 115Q, 116A,E,P,T,Q,117D,E,K 118I,L,M,T, 119G,K,R,S, 120K,S,T,Q, 121A,D,M,P,T,V, 122D,123P,S, 124L,T,V, 136P, 137P, 141C, 154P, 161P, 162C, 164D,E, 167Q,171T, 176C, 177C, 179G,I,K,N,Q, 180A,E,G,T, 181D,G,I,K, 182H,K,S,Q,183A,L,P,S,V,Q, 184*, 185*, 186*, 196Q, 199C, 200K,R, 202N, 203T, 218Q,223E, 239Q, 240P, 241Q, 247C, 273L,Q, 276K,R, 281H, 282P, 283P, 284P,285G,N,R, 286K,Q, 289P, 294T, 299L, 308A, 314G,N, 316D, 324N, 331K,339D, 351Y, 355P, 362K,R, 379K,R, 385D, 406A, 409D,E, 410D,E, and/or411R,K.

The nomenclature used herein for alterations is described in detail inthe section “Alterations, such as Substitutions, Deletions, Insertions.”

Preferably the phytase of the invention comprises at least one of thefollowing alterations: 1*, 2*, 3*, 4P, 5P, 31C, 46E, 52C,E, 53V, 55D,57Y, 59C, 76G, 82E, 99C, 100C, 107D,E,G, 109A, 111P, 114T, 115Q, 116AT,117D, 118T, 119K,R,S, 120S, 121D,P,T, 122D, 123P, 124L, 137P, 141C,161P, 162C, 164E, 167Q, 179K, 180E,T, 181D,K, 182H,K,Q, 183L,V,Q, 184*,185*, 186*, 196Q, 199C, 200K, 202N, 218Q, 223E, 241Q, 273L, 276K,R,285G,R, 286Q, 299L, 314G,N, 331K, 339D, 362K,R, 379K,R, 385D, 406A,410D,E, and/or 411R,K; and/or wherein the amino acids in position 179,180, 181, 182, 183, 184, 185, and 186 have been replaced by KEKHQ (SEQID NO: 21), KEKQQ (SEQ ID NO: 22), KEKKV (SEQ ID NO: 23), or KTDKL (SEQID NO: 24).

In another preferred embodiment, the amino acids in position 179, 180,181, 182, 183, 184, 185, and 186 have been replaced by QADKP (SEQ ID NO:17), GEDKP (SEQ ID NO: 18), NGISA (SEQ ID NO: 19), IAGKS (SEQ ID NO:20), KEKHQ (SEQ ID NO: 21), KEKQQ (SEQ ID NO: 22), KEKKV (SEQ ID NO:23), or KTDKL (SEQ ID NO: 24).

The invention also relates to a phytase which has at least 74% identityto SEQ ID NO: 2 and which comprises at least one of the followingalterations: 1H,K,R, 60P, 105E, 106A,G, 155F, 157F, 173P, 175L, 188P,205P, 215M, 231P, 254Y, 280P, 330D, and/or 371P; with the proviso thatthe phytase is not SEQ ID NO: 3, not SEQ ID NO: 4, not SEQ ID NO: 6, andnot SEQ ID NO: 9 and the variants thereof listed in FIG. 1. In apreferred embodiment the phytase comprises the alteration 1K. Inadditional preferred embodiments, the phytase comprises the followingcombinations of alterations: 280P/282P/283P, 155F/254Y, and/or155F/157F/254Y.

Preferred phytases of the invention comprise an alteration selected fromthe following: 52C, 141C, 162C, 31C, 52C, 99C, 59C, 100C, 141C/199C, 4P,5P, 111P, 137P, 161P, 52E, 57Y, 76G, 107D, 107G, 109A, 1*, 1*/2*,1*/2*/3*, 121T, 273L, 285G, 286Q, 299L, 362K, 331K/55D, 107E, 46E, 82E,119R, 119K, 164E, 223E, 276R, 276K, 362R, 379R, 379K, 385D, 410D, 410E,411R, 411K, 53V, 121D, 167Q, 196Q, 200K, 202N, 218Q, 241Q, 285N, 314N,314G, 406A, 179K/180E/181K/182H/183Q/184*/185*/186*,179K/180E/181K/182Q/183Q/184*/185*/186*,179K/180E/181K/182K/183V/184*/185*/186*,179K/180T/181D/182K/183L/184*/185*/186*, 111P/241Q, 1K,114T/115Q/116A/117D/118T/119S/120S/121P/122D/123P/124L, and114T/115Q/116T/117D/118T/119S/120S/121P/122D/123P/124L.

The phytase of the invention may be a variant of any wildtype or variantphytase. In particular embodiments, it is a variant of the phytase ofSEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 9, ora variant of any one of the phytase variants related to SEQ ID NO: 9 andlisted in FIG. 1.

The phytase of the invention may furthermore comprise an alteration(substitution) or a combination of alterations (substitutions) selectedfrom amongst the alterations (substitutions) and combinations ofalterations (substitutions) listed in each row of FIG. 1.

Particularly preferred variants of the phytase of SEQ ID NO: 2 are thefollowing: R339D, N4P, GSP, Q111P, E1*, E1*/E2*, E1*/E2*/Q3*, M273L, andN286K; as well as any combination thereof; as well as the correspondingvariants of SEQ ID NO: 3, 4 and 6.

Particularly preferred phytases of the invention comprise at least oneof the following alterations: 339D, 4P, 5P, 111P, 1*, 1*/2*, 1*/2*/3*,273L, and/or 286K.

The invention also relates to a phytase which has at least 74% identityto SEQ ID NO: 2 and which comprises at least one of the followingalterations:

(i) 141C/199C, 910/460, 52C/99C, 31C/176C, 31C/177C, 59C/100C, and/or162C/247C;

(ii) 41P, 91P, 136P, 137P, 154P, 161P, 355P, 111P, 240P, 282P, 283P,284P, 289P, 4P, and/or 5P;

(iii) 52E, 55I, 57Y, 104A/105F, 107D,G, 109A,G, 76G, 84Y, 121T, 362K,273L,Q, 285G,R, 286K,Q, 294T, 299L, 331K/55D, and/or 351Y;

(iv) 1*, 1*/2*, or 1*/2*/3*;

(v) wherein K179, T180, T181, E182, K183, S184, T185, and K186 have beenreplaced by QADKP (SEQ ID NO: 17), GEDKP (SEQ ID NO: 18), NGISA (SEQ IDNO: 19), IAGKS (SEQ ID NO: 20), KEKHQ (SEQ ID NO: 21), KEKQQ (SEQ ID NO:22), KEKKV (SEQ ID NO: 23), or KTDKL (SEQ ID NO: 24);(vi) 119R,K, and/or 411R,K;(vii) 107E, and/or 164E,D;(viii) 362R,K, 276R,K, 379R,K, 409D,E, 223E, 385D, 46D,E, 410D,E, and/or82E;(ix) 218Q, 324N, 200R,K, 121D, 196Q, 202N, 406A, 167Q, 53V,Q, 241Q,314N,G, 239Q, and/or 285N;(x) 114H/115Q/116E/117K/118M/119G/120T/121M/122D/123P/124T,114H/115Q/116Q/117D/118I/119K/120Q/121V/122D/123S/124L,114H/115Q/116P/117E/118I/119G/120K/121M/122D/123P/124V,114T/115Q/116A/117D/118T/119S/120S/121P/122D/123P/124L,114H/115Q/116Q/117D/118I/119K/120Q/121A/122D/123P/124L,114T/115Q/116T/117D/118T/119S/120S/121P/122D/123P/124L, or114N/115Q/116A/117D/118L/119K/120K/121T/122D/123P/124L;(xi) 31T, 74A, 171T, 203T, 281H, 316D, and/or 308A; and/or(xii) 339D.Strategy for Preparing Variants

The structure of the C. braakii ATCC 51113 phytase was built by homologymodelling, using as a template the structure of the E. coli AppA phytase(Protein Data Bank id.: 1 DKO; Lim et al., 2000, Nat. Struct. Biol. 2:108-113).

The structure was subjected to molecular dynamics (MD) simulations andelectrostatic calculations. Positions for putative disulfide bridges andprolines were also identified, as well as other positions of potentialimportance as regards the various desirable enzymatic properties.Finally, putative glycosylation sites (stretches of NXT or NXS) wereidentified.

All these suggestions were evaluated within the framework of themodelled structure and the simulation results, for the thermostabilityproperty with particular emphasis at the high temperature end.

The corresponding phytase variants were prepared by methods known in theart and tested as described in the experimental part.

Phytase Polypeptides, Percentage of Identity

In the present context a phytase is a polypeptide having phytaseactivity, i.e., an enzyme which catalyzes the hydrolysis of phytate(myo-inositol hexakisphosphate) to (1) myo-inositol and/or (2) mono-,di-, tri-, tetra- and/or penta-phosphates thereof and (3) inorganicphosphate.

In the present context the term a phytase substrate encompasses, i.a.,phytic acid and any phytate (salt of phytic acid), as well as thephosphates listed under (2) above.

The ENZYME site at the internet (www.expasy.ch/enzyme/) is a repositoryof information relative to the nomenclature of enzymes. It is primarilybased on the recommendations of the Nomenclature Committee of theInternational Union of Biochemistry and Molecular Biology (IUB-MB) andit describes each type of characterized enzyme for which an EC (EnzymeCommission) number has been provided (Bairoch A. The ENZYME database,2000, Nucleic Acids Res. 28:304-305). See also the handbook EnzymeNomenclature from NC-IUBMB, 1992).

According to the ENZYME site, three different types of phytases areknown: A so-called 3-phytase (alternative name 1-phytase; a myo-inositolhexaphosphate 3-phosphohydrolase, EC 3.1.3.8), a so-called 4-phytase(alternative name 6-phytase, name based on 1L-numbering system and not1D-numbering, EC 3.1.3.26), and a so-called 5-phytase (EC 3.1.3.72). Forthe purposes of the present invention, all three types are included inthe definition of phytase.

In a particular embodiment, the phytases of the invention belong to thefamily of acid histidine phosphatases, which includes the Escherichiacoli pH 2.5 acid phosphatase (gene appA) as well as fungal phytases suchas Aspergillus awamorii phytases A and B (EC: 3.1.3.8) (gene phyA andphyB). The histidine acid phosphatases share two regions of sequencesimilarity, each centered around a conserved histidine residue. Thesetwo histidines seem to be involved in the enzymes' catalytic mechanism.The first histidine is located in the N-terminal section and forms aphosphor-histidine intermediate while the second is located in theC-terminal section and possibly acts as proton donor.

In a further particular embodiment, the phytases of the invention have aconserved active site motif, viz. R-H-G-X-R-X-P, wherein X designatesany amino acid (see amino acids 16 to 22 of SEQ ID NOs:2, 3, 4, 6 andamino acids 38-44 of SEQ ID NO: 9). In a preferred embodiment, theconserved active site motif is R-H-G-V-R-A-P, i.e., amino acids 16-22(by reference to SEQ ID NO: 2) are RHGVRAP.

For the purposes of the present invention the phytase activity isdetermined in the unit of FYT, one FYT being the amount of enzyme thatliberates 1 micro-mol inorganic ortho-phosphate per min. under thefollowing conditions: pH 5.5; temperature 37° C.; substrate: sodiumphytate (C₆ H₆O₂₄P₆Na₁₂) in a concentration of 0.0050 mol/l. Suitablephytase assays are the FYT and FTU assays described in Example 1 of WO00/20569. FTU is for determining phytase activity in feed and premix.Phytase activity may also be determined using the assays of Example 1(“Determination of phosphatase activity” or “Determination of phytaseactivity”).

In a particular embodiment the phytase of the invention is isolated. Theterm “isolated” as used herein refers to a polypeptide which is at least20% pure, preferably at least 40% pure, more preferably at least 60%pure, even more preferably at least 80% pure, most preferably at least90% pure, and even most preferably at least 95% pure, as determined bySDS-PAGE. In particular, it is preferred that the polypeptides are in“essentially pure form”, i.e., that the polypeptide preparation isessentially free of other polypeptide material with which it is nativelyassociated. This can be accomplished, for example, by preparing thepolypeptide by means of well-known recombinant methods or by classicalpurification methods.

The relatedness between two amino acid sequences is described by theparameter “identity”. For purposes of the present invention, thealignment of two amino acid sequences is determined by using the Needleprogram from the EMBOSS package (http://emboss.org) version 2.8.0. TheNeedle program implements the global alignment algorithm described inNeedleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453. The substitutionmatrix used is BLOSUM62, gap opening penalty is 10, and gap extensionpenalty is 0.5.

The degree of identity between an amino acid sequence of the presentinvention (“invention sequence”) and the amino acid sequence referred toin the claims (SEQ ID NO: 2) is calculated as the number of exactmatches in an alignment of the two sequences, divided by the length ofthe “invention sequence,” or the length of the SEQ ID NO: 2, whicheveris the shortest. The result is expressed in percent identity.

An exact match occurs when the “invention sequence” and SEQ ID NO: 2have identical amino acid residues in the same positions of the overlap(in the alignment example below this is represented by “|”). The lengthof a sequence is the number of amino acid residues in the sequence(e.g., the length of amino acids 1-411 of SEQ ID NO: 2 is 411).

Example 13 is an example of an alignment of the phytase of SEQ ID NO: 2and the phytase of SEQ ID NO: 9, and the example illustrates how tocalculate the percentage of identity between these two backbones.

In another, purely hypothetical, alignment example below, the overlap isthe amino acid sequence “HTWGER-NL” of Sequence 1; or the amino acidsequence “HGWGEDANL” of Sequence 2. In the example a gap is indicated bya “-”.

Hypothetical alignment example:

Sequence 1: ACMSHTWGER-NL                 | |||  ||Sequence 2:     HGWGEDANLAMNPS

In a particular embodiment, the percentage of identity of an amino acidsequence of a polypeptide with, or to, SEQ ID NO: 2 is determined by i)aligning the two amino acid sequences using the Needle program, with theBLOSUM62 substitution matrix, a gap opening penalty of 10, and a gapextension penalty of 0.5; ii) counting the number of exact matches inthe alignment; iii) dividing the number of exact matches by the lengthof the shortest of the two amino acid sequences, and iv) converting theresult of the division of iii) into percentage.

In the above hypothetical example, the number of exact matches is 6, thelength of the shortest one of the two amino acid sequences is 12;accordingly the percentage of identity is 50%.

In particular embodiments of the phytase of the invention, the degree ofidentity to SEQ ID NO: 2 is at least 75%, 76%, 77%, 78%, 79%, 80%, 81%,82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, or at least 99%. In still further particular embodiments,the degree of identity is at least 98.0%, 98.2%, 98.4%, 98.6%, 98.8%,99.0%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or atleast 99.9%. In alternative embodiments, the degree of identity is atleast 70%, 71%, 72%, or at least 73%.

In still further particular embodiments, the phytase of the inventionhas no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, or no more than 10alterations as compared to SEQ ID NO: 2; no more than 11, 12, 13, 14,15, 16, 17, 18, 19, or no more than 20 alterations as compared to SEQ IDNO: 2; no more than 21, 22, 23, 24, 25, 26, 27, 28, 29, or no more than30 alterations as compared to SEQ ID NO: 2; no more than 31, 32, 33, 34,35, 36, 37, 38, 39, or not more than 40 alterations as compared to SEQID NO: 2; no more than 41, 42, 43, 44, 45, 46, 47, 48, 49, or no morethan 50 alterations as compared to SEQ ID NO: 2; no more than 51, 52,53, 54, 55, 56, 57, 58, 59, or no more than 60 alterations as comparedto SEQ ID NO: 2; no more than 61, 62, 63, 64, 65, 66, 67, 68, 69, or nomore than 70 alterations as compared to SEQ ID NO: 2; no more than 71,72, 73, 74, 75, 76, 77, 78, 79, or no more than 80 alterations ascompared to SEQ ID NO: 2; no more than 81, 82, 83, 84, 85, 86, 87, 88,89, or no more than 90 alterations as compared to SEQ ID NO: 2; no morethan 91, 92, 93, 94, 95, 96, 97, 98, 99, or no more than 100 alterationsas compared to SEQ ID NO: 2; no more than 101, 102, 103, 104, 105, 106,107, 108, 109, or no more than 110 alterations as compared to SEQ ID NO:2; no more than 111, 112, 113, 114, 115, 116, 117, 118, 119, or no morethan 120 alterations as compared to SEQ ID NO: 2; or no more than 121,122, 123, or 124 alterations as compared to SEQ ID NO: 2.

Position Numbering

The nomenclature used herein for defining amino acid positions is basedon the amino acid sequence of the phytase derived from Citrobacterbraakii ATCC 51113, the mature sequence of which is given in thesequence listing as SEQ ID NO: 2 (amino acids 1-411 of SEQ ID NO: 2).Accordingly, in the present context, the basis for numbering positionsis SEQ ID NO: 2 starting with E1 and ending with E411.

When used herein the term “mature” part (or sequence) refers to thatpart of the polypeptide which is secreted by a cell which contains, aspart of its genetic equipment, a polynucleotide encoding thepolypeptide. In other words, the mature polypeptide part refers to thatpart of the polypeptide which remains after the signal peptide part, aswell as a propeptide part, if any, has been cleaved off. The signalpeptide part can be predicted by programs known in the art (e.g.,SignalP). The expected signal peptide part of SEQ ID NO: 2 is includedin the present sequence listing as SEQ ID NO: 8, which is encoded by SEQID NO: 7. SEQ ID NO: 2 is the expected mature part. Generally, the firstamino acid of the mature part of an enzyme can be determined byN-terminal sequencing of the purified enzyme. Any difference between thesignal peptide part and the mature part must then be due to the presenceof a propeptide.

Alterations, Such as Substitutions, Deletions, Insertions

A phytase variant can comprise various types of alterations relative toa template (i.e., a reference or comparative amino acid sequence such asSEQ ID NO: 2): An amino acid can be substituted with another amino acid;an amino acid can be deleted; an amino acid can be inserted; as well asany combination of any number of such alterations. In the presentcontext the term “insertion” is intended to cover also N- and/orC-terminal extensions.

The general nomenclature used herein for a single alteration is thefollowing: XDcY, where “X” and “Y” independently designate a one-letteramino acid code, or a “*” (deletion of an amino acid), “D” designates anumber, and “c” designates an alphabetical counter (a, b, c, and soforth), which is only present in insertions. Reference is made to Table1 below which describes purely hypothetical examples of applying thisnomenclature to various types of alterations.

TABLE 1 Type Description Example Sub- X = Amino acid in template G80Astitution D = Position in template        80 c emptyAALNNSIGVLGVAPSAELYAVKVLGASGSG Y = Amino acid in variant|||||||:|||||||||||||||||||||| AALNNSIAVLGVAPSAELYAVKVLGASGSG(SEQ ID NOS: 25 and 26) Insertion X = ″*″ *80aT *80bY *85aS D =Position in template        80     85 before the insertionAALNNSIG..VLGVA.PSAELYAVKVLGASG c = ″a″for first insertion at |||||||| ||||| ||||||||||||||| this position, ″b″ for next,AALNNSIGTYVLGVASPSAELYAVKVLGASG etc (SEQ ID NOS: 27 and 28) Deletion X =Amino acid in template V81* D = Position in template        80 c emptyAALNNSIGVLGVAPSAELYAVKVLGASGSG Y = ″*″ |||||||| |||||||||||||||||||||AALNNSIG.LGVAPSAELYAVKVLGASGSG (SEQ ID NOS: 29 and 30) N-terminalInsertions at position ″0″. *0aA *0bT *0cG extension    1...AQSVPWGISRVQ    |||||||||||| ATGAQSVPWGISRVQ (SEQ ID NOS: 31 and 32)C-terminal Insertions after the N- *275aS *275bT extensionterminal amino acid.                 270  275 ATSLGSTNLYGSGLVNAEAATR..|||||||||||||||||||||| ATSLGSTNLYGSGLVNAEAATRST (SEQ ID NOS: 33 and 34)

As explained above, the position number (“D”) is counted from the firstamino acid residue of SEQ ID NO: 2.

Several alterations in the same sequence are separated by “/” (slash),e.g., the designation “1*/2*/3*” means that the amino acids in positionnumbers 1, 2, and 3 are all deleted, and the designation “104A/105F”means that the amino acid in position number 104 is substituted by A,and the amino acid in position number 105 is substituted by F.

Alternative alterations are separated by “,” (comma), e.g., thedesignation “119R,K” means that the amino acid in position 119 issubstituted with R or K.

The commas used herein in various other enumerations of possibilitiesmean what they usually do grammatically, viz. often and/or. E.g., thefirst comma in the listing “53V,Q, 121D, and/or 167Q” denotes analternative (V or Q), whereas the two next commas should be interpretedas and/or options: 53 V or Q, and/or 121D, and/or 167Q.

In the present context, “at least one” (e.g., alteration) means one ormore, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 alterations; or 12, 14, 15,16, 18, 20, 22, 24, 25, 28, or 30 alterations; and so on, up to amaximum number of alterations of 125, 130, 140, 150, 160, 170, 180, 190,or of 200. The phytase variants of the invention, however, still have tobe at least 74% identical to SEQ ID NO: 2, this percentage beingdetermined as described above.

A substitution or extension without any indication of what to substituteor extend with refers to the insertion of any natural, or non-natural,amino acid, except the one that occupies this position in the template.

Example 13 provides further illustration of how to apply thisnomenclature.

Identifying Corresponding Position Numbers

As explained above, the mature phytase of Citrobacter braakii ATCC 51113(SEQ ID NO: 2) is used as the standard for position numbering and,thereby, also for the nomenclature.

For another phytase, in particular a phytase variant of the invention,the position corresponding to position D in SEQ ID NO: 2 is found byaligning the two sequences as specified above in the section entitled“Phytase polypeptides, percentage of identity”. From the alignment, theposition in the sequence of the invention corresponding to position D ofSEQ ID NO: 2 can be clearly and unambiguously identified (the twopositions on top of each other in the alignment).

Example 13 is an example of an alignment of the phytase of SEQ ID NO: 2and the phytase of SEQ ID NO: 9, and the example illustrates howcorresponding positions in these two backbones are identified.

Below some additional, purely hypothetical, examples are included whichare derived from Table 1 above which in the third column includes anumber of alignments of two sequences:

Consider the third cell in the first row of Table 1: The upper sequenceis the template, the lower the variant. Position number 80 refers toamino acid residue G in the template. Amino acid A occupies thecorresponding position in the variant. Accordingly, this substitution isdesignated G80A.

Consider now the third cell in the second row of Table 1: The uppersequence is again the template and the lower the variant. Positionnumber 80 again refers to amino acid residue G in the template. Thevariant has two insertions, viz. TY, after G80 and before V81 in thetemplate. Whereas the T and Y of course would have their own “real”position number in the variant amino acid sequence, for the presentpurposes we always refer to the template position numbers, andaccordingly the T and the Y are said to be in position number 80a and80b, respectively.

Finally, consider the third cell in the last row of Table 1: Positionnumber 275 refers to the last amino acid of the template. A C-terminalextension of ST are said to be in position number 275a and 275b,respectively, although, again, of course they have their own “real”position number in the variant amino acid sequence.

Amended Properties, Reference Phytase

In a particular embodiment, the phytase of the invention has amended,preferably improved, properties. The terms “amended” and “improved”imply a comparison with another phytase. Examples of such other,reference, or comparative, phytases are: SEQ ID NO: 3, and/or SEQ ID NO:4. Still further examples of reference phytases may be SEQ ID NO: 2,and/or SEQ ID NO: 6. A still further example of a reference phytase maybe SEQ ID NO: 9, and the variants thereof disclosed in FIG. 1.

Non-limiting examples of properties that are amended, preferablyimproved, are the following: Thermostability, pH profile, specificactivity, performance in animal feed, protease-sensibility, and/orglycosylation pattern. The phytase of the invention may also have anamended, preferably improved, temperature profile, and/or it mayincorporate a change of a potential protease cleavage site.

Thermostability

Thermostability, or temperature stability, may be determined asdescribed in Example 1 under the heading of “Determination oftemperature stability.” Accordingly, in a preferred embodiment, aphytase of the invention has a residual activity which is higher thanthe residual activity of a reference phytase, wherein residual activityis determined as follows: A fermentation supernatant is divided in twoparts, one part is incubated for 30 minutes at a desired elevatedtemperature, and the other part for 30 minutes at 5° C., following whichthe activity of both is determined on p-nitrophenyl phosphate at 37° C.and pH 5.5, and the activity of the sample having been incubated at anelevated temperature is divided by the activity of the same samplehaving been incubated at 5° C. Preferred elevated temperatures are 50°C., 55° C., 60° C., 65° C., 70° C., 75° C., 80° C., or 85° C. Ifdesired, the enzyme-containing samples may be diluted in 0.1 M NaAc pH5.5. The residual activity of a phytase of the invention is preferablyat least 105%, or at least 110%, 120%, 130%, 140%, 150% of the residualactivity of the reference phytase. In still further embodiments, theresidual activity of a phytase of the invention is at least 200%, or atleast 250%, 300%, 400%, or at least 500% of the residual activity of thereference phytase. In still further embodiments, the residual activityof a phytase of the invention is at least 2×, 3×, 4×, 5×, 6×, 7×, 10×,15×, 20×, or at least 25× the residual activity of the referencephytase.

Thermostability may also be determined as described in Example 5.Accordingly, in a preferred embodiment, a phytase of the invention has aresidual activity which is higher than the residual activity of areference phytase, wherein residual activity is determined as follows: Afermentation supernatant is divided in two parts, one part is incubatedfor 30 minutes at a 50° C., and the other part for 30 minutes at 5° C.,following which the activity of both is determined on p-nitrophenylphosphate at 37° C. and pH 5.5, and the activity of the sample havingbeen incubated at an elevated temperature is divided by the activity ofthe same sample having been incubated at 5° C. If desired, theenzyme-containing samples may be diluted in 0.1 M NaAc pH 5.5. Theresidual activity of a phytase of the invention is preferably at least2×, 3×, 4×, 5×, 6×, 7×, 10×, 15×, 20×, or at least 25× the residualactivity of the reference phytase of SEQ ID NO: 3. The residual activityof a phytase of the invention is preferably at least 105%, or at least110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, or at least 200%of the residual activity of the reference phytase of SEQ ID NO: 2. Thefollowing substitutions are particularly preferred as they improvethermostability as compared to the phytase of SEQ ID NO: 3 as well as tothe phytase of SEQ ID NO: 2 (see Table 3): 4P, 5P, 111P, 1*, 1*/2*,1*/2*/3*, 273L, and/or 286Q.

Thermostability may also be determined as described in Example 8.Accordingly, in a preferred embodiment, a phytase of the invention has aresidual activity which is higher than the residual activity of areference phytase, wherein residual activity is determined as follows: Afermentation supernatant is divided in two parts, one part is incubatedfor 30 minutes at a 60° C., and the other part for 30 minutes at 5° C.,following which the activity of both is determined on p-nitrophenylphosphate at 37° C. and pH 5.5, and the activity of the sample havingbeen incubated at an elevated temperature is divided by the activity ofthe same sample having been incubated at 5° C. If desired, theenzyme-containing samples may be diluted in 0.1 M NaAc pH 5.5,optionally including 0.005% Tween-20. The phytase of the invention andthe reference phytase may be expressed in a Bacillus subtilis hoststrain. The host strain may be grown in 100 ml PS1 medium (100 g/Lsucrose, 40 g/L Soy flakes, 10 g/L Na₂HPO₄.12H₂O, 0.1 ml/L Dowfax 63N10(Dow)) in 500 ml shake flasks for four days at 30° C. at 300 rpm. Theresidual activity of a phytase of the invention is preferably at least32%, or at least 34%, 36%, 38%, or at least 40% of the residual activityof the reference phytase of SEQ ID NO: 2. More preferably, the residualactivity of a phytase of the invention is at least 50%, or at least 60%,70%, 80%, 90%, or at least 100% of the residual activity of thereference phytase of SEQ ID NO: 2. Even more preferably the residualactivity of a phytase of the invention is at least 120%, 140%, 160%,180%, or at least 200% of the residual activity of the reference phytaseof SEQ ID NO: 2. Most preferably, the residual activity of a phytase ofthe invention is at least 2×, or at least 3×, 4×, or at least 5× theresidual activity of the reference phytase of SEQ ID NO: 2. Thefollowing substitutions are particularly preferred (see Table 5):

(i) 409E, 136P;

(ii) 411K, 331K/55D, 167Q, 179K/180T/181D/182K/183L/184*/185*/186*,107E;

(iii) 196Q, 276R, 285G, 299L, 200K;

(iv) 119R, 121 D, 107D, 179K/180E/181K/182H/183Q/184*/185*/186*;

(v) 314N, 161P, 410D, 141C, 179K/180E/181K/182Q/183Q/184*/185*/186*,285N;

(vi) 164E, 411R, 52C, 137P, 314G;

(vii) 1K, 1*/2*/3*, 121T, 406A, 82E, 109A;

(viii) 5P, 57Y, 379R, 1*/2*;

(ix) 410E, 1*, 119K, 52E;

(x) 4P, 362K, 202N, 276K, 385D;

(xi) 111P/241Q, 162C, 179K/180E/181K/182K/183V/184*/185*/186*, 241Q;

(xii) 223E, 286Q, 107G,114T/115Q/116A/117D/118T/119S/120S/121P/122D/123P/124L, 379K, 273L;

(xiii) 31C, 53V, 59C/100C;

(xiv) 46E, 111P, 114T/115Q/116T/117D/118T/119S/120S/121P/122D/123P/124L,76G, 362R;

(xv) 141C/199C, 52C/99C.

Thermostability may also be determined as described in Example 9, i.e.,using DSC measurements to determine the denaturation temperature, Td, ofthe purified phytase protein. The Td is indicative of thethermostability of the protein: The higher the Td, the higher thethermostability. Accordingly, in a preferred embodiment, the phytase ofthe invention has a Td which is higher than the Td of a referencephytase, wherein Td is determined on purified phytase samples(preferably with a purity of at least 95%, determined by SDS-PAGE),after dialysis in 20 mM Na-acetate pH4.0 (preferably in a 2-3 h stepfollowed by an over night step), followed by 0.45 um filtration anddilution with dialysis buffer to a protein concentration correspondingto approximately 2 absorbancy units (A₂₈₀), using Differential Scanningcalorimetry at a 90° C./h scan rate from 20-90° C. in 20 mM Na-acetatebuffer, pH 4.0. In a preferred embodiment, the Td of the phytase of theinvention is higher than the Td of the phytase of SEQ ID NO: 4, morepreferably at least 101% thereof, or at least 102%, 103%, 104%, 105%,106%, 107%, 108%, 109%, or at least 110% thereof. Even more preferably,the Td of the phytase of the invention is at least 120%, 130%, 140%,150%, 160%, 170%, 180%, or at least 190% of the Td of the phytase of SEQID NO: 4. The following substitutions are particularly preferred (seeTable 6): 362K, 362R, 111P, and/or 273L. In still further particularembodiments, the thermostable phytase of the invention has a meltingtemperature, Tm (or a denaturation temperature, Td), as determined usingDifferential Scanning calorimetry (DSC) as described in Example 2 (i.e.,in 20 mM sodium acetate, pH 4.0), of at least 50° C. In still furtherparticular embodiments, the Tm is at least 51, 52, 53, 54, 55, 56, 57,58, 59, 60, 61, 62, 62.5. 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73,74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91,92, 93, 94, 95, 96, 97, 98, 99 or at least 100° C. DSC measurements mayalso be performed as described in Example 1 (“DSC measurements”), orExample 2 (“Thermostability by DSC”).

Thermostability may also be determined as described in Example 12.Accordingly, in a preferred embodiment the phytase of the invention,after incubation for 60 minutes at 70° C. and pH 4.0, has an improvedresidual activity as compared to the residual activity of a referencephytase treated in the same way, the residual activity being calculatedfor each phytase relative to the activity found before the incubation(at 0 minutes). The residual activity is preferably measured on sodiumphytate at pH 5.5 and 37° C. The incubation is preferably in 0.1 Msodium acetate, pH 4.0. The phytase is preferably purified, morepreferably to a purity of at least 95%, determined by SDS-PAGE. Apreferred phytase activity assay buffer is 0.25 M Na-acetate pH 5.5.Using this method, the residual activity of the phytase of the inventionis preferably at least 105% of the residual activity of the referencephytase, more preferably at least 110%, 115%, 120%, 130%, 140%, 150%,160%, 170%, 180%, 190%, or at least 200%. In the alternative, theresidual activity relative to the activity at 0 minutes is preferably atleast 31%, or at least 32%. The following substitutions providingimproved thermostability stability are preferred (see Table 9): 273L,46E, 362R, and/or 53V.

In a particular embodiment, the phytase variant of the invention is morethermostable than the reference phytase, wherein thermostability isdetermined using any of the above-mentioned four tests (based on Example1, 5, 8, 9, or 12).

In particular embodiments, an improved thermostability is expected ofthe following variants of the phytase of SEQ ID NO: 2 (in order ofpreference, within each grouping):

(i) K141C/V199C, Q91C/W46C, G52C/A99C, N31C/E176C, N31C/T177C,G59C/F100C, S162C/S247C;

(ii) D41P, Q91P, N136P, T137P, L154P, S161P, T355P, Q111P, K240P, G282P,T283P, T284P, G289P, N4P, G5P;

(iii) G52E, V55I, E57Y, L104A/A105F, K107D,G, Q109A,G, T76G, A84Y,N121T, 1362K, M273L,Q, E285G,R, N286Q, V294T, 1299L, E331K/V55D, F351Y;

(iv) E1*, E1*/E2*, E1*/E2*/Q3*;

(v) replacing the loop comprised between C178 and C187 with shorterloops selected from, e.g., QADKP (SEQ ID NO: 17), GEDKP (SEQ ID NO: 18),NGISA (SEQ ID NO: 19), IAGKS (SEQ ID NO: 20), KEKHQ (SEQ ID NO: 21),KEKQQ (SEQ ID NO: 22), KEKKV (SEQ ID NO: 23), KTDKL (SEQ ID NO: 24);(vi) E119R,K, E411R,K;(vii) K107E, R164E,D;(iix) 1362R,K, T276R,K, I379R,K, V409D,E, Q223E, N385D, W46D,E, T410D,E,Q82E.(ix) replacing the loop between residues 114 and 124 (YQKDEEKNDPL) whichfaces the active site with a loop selected from, e.g., HQEKMGTMDPT (SEQID NO: 10), HQQDIKQVDSL (SEQ ID NO: 11), HQPEIGKMDPV (SEQ ID NO: 12),TQADTSSPDPL (SEQ ID NO: 13), HQQDIKQADPL (SEQ ID NO: 14), TQTDTSSPDPL(SEQ ID NO: 15), NQADLKKTDPL (SEQ ID NO: 16);(x) R339D.Temperature Profile

Whether or not a phytase of the invention has an amended temperatureprofile as compared to a reference phytase may be determined asdescribed in Example 10. Accordingly, in a particular embodiment thephytase of the invention has an amended temperature profile as comparedto a reference phytase, wherein the temperature profile is determined asphytase activity as a function of temperature on sodium phytate at pH5.5 in the temperature range of 20-90° C. (in 10° C. steps). A preferredbuffer is in 0.25 M Na-acetate buffer pH 5.5. The activity at eachtemperature is preferably indicated as relative activity (in %)normalized to the value at optimum temperature. The optimum temperatureis that temperature within the tested temperatures (i.e., those with 10°C. jumps) where the activity is highest.

In a preferred embodiment, the phytase of the invention has a relativeactivity at 70° C. of at least 18%, or at least 19%, 20%, 21%, 22%, 23%,24%, or at least 25%. As explained above, this is relative to theactivity at the optimum temperature. More preferably, the phytase of theinvention has a relative activity at 70° C. of at least 26%, 27%, 28%,29%, 30%, 31%, or at least 32%. Preferred substitutions which provide anamended temperature profile (in the form of a higher relative activityat 70° C.) are (see Table 7): 57Y, 76G, 107G, 273L, 362K, 46E, 362R,53V, and/or 241Q. Their relative activity at 70° C. is higher ascompared to the reference phytase of SEQ ID NO: 3 and 4, and in someinstances (57Y, 76G, 107G, 273L, 362K, 362R, and/or 53V) also ascompared to the reference phytase of SEQ ID NO: 2.

pH Profile

Whether or not a phytase of the invention has an amended pH profile ascompared to a reference phytase may be determined as described inExample 11. Accordingly, in a particular embodiment the phytase of theinvention has an amended pH profile as compared to a reference phytase,wherein the pH profile is determined as phytase activity as a functionof pH on sodium phytate at 37° C. in the pH range of 2.0 to 7.5 (in 0.5pH-unit steps). A preferred buffer is a cocktail of 50 mM glycine, 50 mMacetic acid and 50 mM Bis-Tris. Another preferred buffer is 0.25 Msodium acetate. The activity at each pH is preferably indicated asrelative activity (in %) normalized to the value at optimum pH.

An example of an amended pH profile is where the pH curve (relativeactivity as a function of pH) is shifted towards higher, or lower, pH.Preferred substitutions which provide a shift of 0.5 pH units towards ahigher pH as compared to the reference phytase of SEQ ID NO: 2, 3 or 4are (see Table 8): 46E, and/or 218Q.

Another example of an amended pH profile is where the optimum pH ischanged, in the upward or the downward direction. Preferredsubstitutions which provide a lower optimum pH as compared to SEQ ID NO:2, 3, and 4 are (see Table 8): 46E, 121D, and/or 200K. Preferredsubstitutions which provide a higher optimum pH as compared to SEQ IDNO: 2, 3, and 4 are (see Table 8): 218Q, and/or 241Q.

An amended pH profile may also be determined as described in Example 1(“Amended pH profile: Determination of pH 3.5/5.5 activity ratio”), viz.by comparing phosphatase activity at pH 3.5 and 5.5. Alternatively, theactivity at pH 3.5 may be compared with the activity at pH 4.0, 4.5, or5.0. In a still further alternative embodiment, phytase activities arecompared instead of phosphatase activities.

In a particular embodiment, the phytase of the invention has an amendedpH profile as compared to a reference phytase. More in particular, thepH profile is amended in the pH-range of 3.5-5.5. Still more inparticular, the activity at pH 4.0, 4.5, 5.0, and/or 5.5 is at a levelof at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or at least 95%of the activity at the pH-optimum (pH 3.5).

The pH profile, as well as the pH-optimum, of a polypeptide may bedetermined by incubating it at various pH-values, using a substrate in apre-determined concentration and a fixed incubation temperature. The pHprofile is a graphical representation of phytase activity versus pH, thepH-optimum is determined from the pH profile. In a particularembodiment, the phosphatase or phytase assay of Example 1 is used, e.g.,the substrate is 5 mM sodium phytate, the reaction temperature 37° C.,and the activity is determined at various pH-values, for example pH2-12, replacing the pH 5.5 acetate buffer with a suitable buffer.Examples of suitable buffers are: 0.1 M glycine/HCl (pH 2.0-3.5), 0.1 MNaAc/Ac (pH 4.0-5.0), 0.1 M Bis-Tris/HCl (pH 5.5-6.5), 0.1 M Tris/HCl(pH 7.0). Other examples of buffers are: 100 mM succinic acid, 100 mMHEPES, 100 mM CHES, 100 mM CABS adjusted to pH-values 2.0, 2.5, 3.0,3.5, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0, 11.0, and 12.0 with HCl orNaOH.

In particular embodiments, an amended pH profile is expected of thefollowing variants of the phytase of SEQ ID NO: 2 (in order ofpreference, within each grouping):

(i) E218Q, D324N, T200R,K, N121D, E196Q, D202N, E406A, E167Q, E53V,Q,E241Q, D314N,G, E239Q, E285N;

(ii) replacing the loop between residues 114 and 124 (YQKDEEKNDPL) whichfaces the active site with a loop selected from, e.g., HQEKMGTMDPT (SEQID NO: 10), HQQDIKQVDSL (SEQ ID NO: 11), HQPEIGKMDPV (SEQ ID NO: 12),TQADTSSPDPL (SEQ ID NO: 13), HQQDIKQADPL (SEQ ID NO: 14), TQTDTSSPDPL(SEQ ID NO: 15), NQADLKKTDPL (SEQ ID NO: 16).Specific Activity

In a particular embodiment, the phytase of the invention has an improvedspecific activity relative to a reference phytase. More in particular,the specific activity of a phytase of the invention is at least 105%,relative to the specific activity of a reference phytase determined bythe same procedure. In still further particular embodiments, therelative specific activity is at least 110, 115, 120, 125, 130, 140,145, 150, 160, 170, 180, 190, 200, 220, 240, 260, 280, 300, 350 or even400%, still relative to the specific activity of the reference phytaseas determined by the same procedure.

In the alternative, the term high specific activity refers to a specificactivity of at least 200 FYT/mg Enzyme Protein (EP). In particularembodiments, the specific activity is at least 300, 400, 500, 600, 700,800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900,2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900 or 3000FYT/mg EP.

Specific activity is measured on highly purified samples (an SDS polyacryl amide gel should show the presence of only one component). Theenzyme protein concentration may be determined by amino acid analysis,and the phytase activity in the units of FYT, determined as described inExample 1. Specific activity is a characteristic of the specific phytasevariant in question, and it is calculated as the phytase activitymeasured in FYT units per mg phytase variant enzyme protein. See Example7 for further details.

In particular embodiments, an amended specific activity is expected ofthe following variants of the phytase of SEQ ID NO: 2, in which, inorder of preference, the loop between residues 114 and 124 (YQKDEEKNDPL)which faces the active site is replaced with a loop selected from, e.g.,HQEKMGTMDPT (SEQ ID NO: 10), HQQDIKQVDSL (SEQ ID NO: 11), HQPEIGKMDPV(SEQ ID NO: 12), TQADTSSPDPL (SEQ ID NO: 13), HQQDIKQADPL (SEQ ID NO:14), TQTDTSSPDPL (SEQ ID NO: 15), NQADLKKTDPL (SEQ ID NO: 16).

Performance in Animal Feed

In a particular embodiment the phytase of the invention has an improvedperformance in animal feed as compared to a reference phytase. Theperformance in animal feed may be determined by the in vitro model ofExample 6. Accordingly, in a preferred embodiment the phytase of theinvention has an improved performance in animal feed, wherein theperformance is determined in an in vitro model, by preparing feedsamples composed of 30% soybean meal and 70% maize meal with added CaCl₂to a concentration of 5 g calcium per kg feed; pre-incubating them at40° C. and pH 3.0 for 30 minutes followed by addition of pepsin (3000U/g feed) and phytase; incubating the samples at 40° C. and pH 3.0 for60 minutes followed by pH 4.0 for 30 minutes; stopping the reactions;extracting phytic acid and inositol-phosphates by addition of HCl to afinal concentration of 0.5 M and incubation at 40° C. for 2 hours,followed by one freeze-thaw cycle and 1 hour incubation at 40° C.;separating phytic acid and inositol-phosphates by high performance ionchromatography; determining the amount of residual phytate phosphorus(IP6-P); calculating the difference in residual IP6-P between thephytase-treated and a non-phytase-treated blank sample (this differenceis degraded IP6-P); and expressing the degraded IP6-P of the phytase ofthe invention relative to degraded IP6-P of the reference phytase (e.g.,the phytases having SEQ ID NO: 3 and 4).

The phytase of the invention and the reference phytase are of coursedosed in the same amount, preferably based on phytase activity units(FYT). A preferred dosage is 125 FYT/kg feed. Another preferred dosageis 250 FYT/kg feed. The phytases may be dosed in the form of purifiedphytases, or in the form of fermentation supernatants. Purified phytasespreferably have a purity of at least 95%, as determined by SDS-PAGE.

In preferred embodiments, the degraded IP6-P value of the purifiedphytase of the invention, relative to the degraded IP6-P value of thereference phytase, is at least 101%, or at least 102%, 103%, 104%, 105%,110%, 115%, or at least 120%. In still further preferred embodiments,the degraded IP6-P value of the purified phytase of the invention,relative to the degraded IP6-P value of the reference phytase, is atleast 125%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, or at least 200%.Preferably, the degraded IP6-P value of the phytase of the invention,relative to the degraded IP6-P value of the SEQ ID NO: 2 phytase, is atleast 105%, 110%, 113%, 115%, 120%, 125%, or at least 130%.

The following substitutions provide an improved or at least as goodperformance in animal feed in vitro (see Table 4A) as compared to thephytase of SEQ ID NO: 3: 4P, 5P, 111P, 1*, 1*/2*, 1*/2*/3*, 273L, 286Q.

The following substitutions also provide an improved or at least as goodperformance in animal feed in vitro (see Table 4B) as compared to thephytase of SEQ ID NO: 3: 57Y, 76G, 107G, 362K, 362R, 121D, 196Q, 200K,202N, 314N, 406A, and114T/115Q/116A/117D/118T/119S/120S/121P/122D/123P/124L.

Even more preferred substitutions when it comes to animal feedperformance are: 57Y, 76G, 362K, 362R, 121D, 196Q, 200K, 202N, and 406A.

The relative performance of a phytase of the invention may also becalculated as the percentage of the phosphorous released by thereference phytase.

In a still further particular embodiment, the relative performance ofthe phytase of the invention may be calculated as the percentage of thephosphorous released by the phytase of the invention, relative to theamount of phosphorous released by the reference phytase.

In still further particular embodiments, the relative performance of thephytase of the invention is at least 105%, preferably at least 110, 120,130, 140, 150, 160, 170, 180, 190, or at least 200%.

Reduced Protease-Sensibility

In a particular embodiment, the phytase of the invention has a reducedprotease-sensibility. More in particular, it has a reduced sensibilitytowards the Kex2 protease, meaning a reduced tendency to become cleavedby this protease.

Variant 339D, preferably R339D, is an example of a phytase of theinvention with a reduced protase-sensibility.

Glycosylation Pattern

Glycosylation is a phenomenon which is only observed when expressingproteins in eukaryotes such as fungi and transgenic plants, but not inprokaryotes such as bacteria. There are various types of glycosylation,but in the present context the most relevant is the N-glycosylation,i.e., the asparagine-linked glycosylation where sugars are attached to aprotein, starting from an N-acetyglucosamine molecule attached toasparagines. N-glycosylation has been found to occur only to asparaginesthat in the sequence are part of the following tripeptides: N-X-T orN-X-S, where X designates any amino acid.

Surprisingly, a lower thermostability was observed when the phytase ofSEQ ID NO: 2 was expressed in the fungus (yeast) Pichia pastoris, ascompared to when it was expressed in Bacillus subtilis, see Example 2.

This observation has led to the proposal of the present invention thatthermostability may be improved for phytases expressed in fungi byaltering potential glycosylation sites.

The present invention accordingly also relates to phytase variantshaving an amended glycosylation pattern, preferably amendedN-glycosylation sites. The amended glycosylation is expected to conferan improved thermostability upon the phytase variant, when expressed ina fungus.

Examples of phytases are bacterial phytases, e.g., Gram-negativephytases, such as E. coli and Citrobacter phytases and variants thereof,including the phytases of the present invention as well as the phytasesof SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 6, and SEQ IDNO: 9 herein. Examples of fungal expression hosts are Pichia,Saccharomyces, and Aspergillus species.

In particular embodiments, an amended glycosylation pattern is expectedof the following phytases of the invention (e.g., variants of SEQ ID NO:2), in order of preference: N31T, N74A, N171T, N203T, N281H, N316D,N308A. The following are replacing an N-X-T type pattern: N31T, N74A,N281H. The following are replacing an N-X-S type pattern: N171T, N203T,N308A, N316D.

Low-Allergenic Variants

In a specific embodiment, the phytases of the present invention are(also) low-allergenic variants, designed to invoke a reducedimmunological response when exposed to animals, including man. The termimmunological response is to be understood as any reaction by the immunesystem of an animal exposed to the phytase variant. One type ofimmunological response is an allergic response leading to increasedlevels of IgE in the exposed animal. Low-allergenic variants may beprepared using techniques known in the art. For example the phytasevariant may be conjugated with polymer moieties shielding portions orepitopes of the phytase variant involved in an immunological response.Conjugation with polymers may involve in vitro chemical coupling ofpolymer to the phytase variant, e.g., as described in WO 96/17929, WO98/30682, WO 98/35026, and/or WO 99/00489. Conjugation may in additionor alternatively thereto involve in vivo coupling of polymers to thephytase variant. Such conjugation may be achieved by genetic engineeringof the nucleotide sequence encoding the phytase variant, insertingconsensus sequences encoding additional glycosylation sites in thephytase variant and expressing the phytase variant in a host capable ofglycosylating the phytase variant, see e.g., WO 00/26354. Another way ofproviding low-allergenic variants is genetic engineering of thenucleotide sequence encoding the phytase variant so as to cause thephytase variants to self-oligomerize, effecting that phytase variantmonomers may shield the epitopes of other phytase variant monomers andthereby lowering the antigenicity of the oligomers. Such products andtheir preparation is described e.g., in WO 96/16177. Epitopes involvedin an immunological response may be identified by various methods suchas the phage display method described in WO 00/26230 and WO 01/83559, orthe random approach described in EP 561907. Once an epitope has beenidentified, its amino acid sequence may be altered to produce alteredimmunological properties of the phytase variant by known genemanipulation techniques such as site directed mutagenesis (see e.g., WO00/26230, WO 00/26354 and/or WO 00/22103) and/or conjugation of apolymer may be done in sufficient proximity to the epitope for thepolymer to shield the epitope.

Nucleic Acid Sequences and Constructs

The present invention also relates to nucleic acid sequences comprisinga nucleic acid sequence which encodes a phytase variant of theinvention.

The term “isolated nucleic acid sequence” refers to a nucleic acidsequence which is essentially free of other nucleic acid sequences,e.g., at least about 20% pure, preferably at least about 40% pure, morepreferably at least about 60% pure, even more preferably at least about80% pure, and most preferably at least about 90% pure as determined byagarose electrophoresis. For example, an isolated nucleic acid sequencecan be obtained by standard cloning procedures used in geneticengineering to relocate the nucleic acid sequence from its naturallocation to a different site where it will be reproduced. The cloningprocedures may involve excision and isolation of a desired nucleic acidfragment comprising the nucleic acid sequence encoding the polypeptide,insertion of the fragment into a vector molecule, and incorporation ofthe recombinant vector into a host cell where multiple copies or clonesof the nucleic acid sequence will be replicated. The nucleic acidsequence may be of genomic, cDNA, RNA, semisynthetic, synthetic origin,or any combinations thereof.

The nucleic acid sequences of the invention can be prepared byintroducing at least one mutation into a template phytase codingsequence or a subsequence thereof, wherein the mutant nucleic acidsequence encodes a variant phytase. The introduction of a mutation intothe nucleic acid sequence to exchange one nucleotide for anothernucleotide may be accomplished by any of the methods known in the art,e.g., by site-directed mutagenesis, by random mutagenesis, or by doped,spiked, or localized random mutagenesis.

Random mutagenesis is suitably performed either as localized orregion-specific random mutagenesis in at least three parts of the genetranslating to the amino acid sequence shown in question, or within thewhole gene. When the mutagenesis is performed by the use of anoligonucleotide, the oligonucleotide may be doped or spiked with thethree non-parent nucleotides during the synthesis of the oligonucleotideat the positions which are to be changed. The doping or spiking may beperformed so that codons for unwanted amino acids are avoided. The dopedor spiked oligonucleotide can be incorporated into the DNA encoding thephytase enzyme by any technique, using, e.g., PCR, LCR or any DNApolymerase and ligase as deemed appropriate.

Preferably, the doping is carried out using “constant random doping”, inwhich the percentage of wild-type and mutation in each position ispredefined. Furthermore, the doping may be directed toward a preferencefor the introduction of certain nucleotides, and thereby a preferencefor the introduction of one or more specific amino acid residues. Thedoping may be made, e.g., so as to allow for the introduction of 90%wild type and 10% mutations in each position. An additionalconsideration in the choice of a doping scheme is based on genetic aswell as protein-structural constraints.

The random mutagenesis may be advantageously localized to a part of theparent phytase in question. This may, e.g., be advantageous when certainregions of the enzyme have been identified to be of particularimportance for a given property of the enzyme.

Alternative methods for providing variants of the invention include geneshuffling, e.g., as described in WO 95/22625 or in WO 96/00343, and theconsensus derivation process as described in EP 897985.

Nucleic Acid Constructs

A nucleic acid construct comprises a nucleic acid sequence of thepresent invention operably linked to one or more control sequences whichdirect the expression of the coding sequence in a suitable host cellunder conditions compatible with the control sequences. Expression willbe understood to include any step involved in the production of thepolypeptide including, but not limited to, transcription,post-transcriptional modification, translation, post-translationalmodification, and secretion.

The term “nucleic acid construct” as used herein refers to a nucleicacid molecule, either single- or double-stranded, which is isolated froma naturally occurring gene or which is modified to contain segments ofnucleic acids in a manner that would not otherwise exist in nature. Theterm nucleic acid construct is synonymous with the term “expressioncassette” when the nucleic acid construct contains the control sequencesrequired for expression of a coding sequence of the present invention.

The term “control sequences” is defined herein to include allcomponents, which are necessary or advantageous for the expression of apolynucleotide encoding a polypeptide of the present invention. Eachcontrol sequence may be native or foreign to the nucleotide sequenceencoding the polypeptide. Such control sequences include, but are notlimited to, a leader, polyadenylation sequence, propeptide sequence,promoter, signal peptide sequence, and transcription terminator. At aminimum, the control sequences include a promoter, and transcriptionaland translational stop signals. The control sequences may be providedwith linkers for the purpose of introducing specific restriction sitesfacilitating ligation of the control sequences with the coding region ofthe nucleotide sequence encoding a polypeptide.

The term “operably linked” denotes herein a configuration in which acontrol sequence is placed at an appropriate position relative to thecoding sequence of the polynucleotide sequence such that the controlsequence directs the expression of the coding sequence of a polypeptide.

When used herein the term “coding sequence” (CDS) means a nucleotidesequence, which directly specifies the amino acid sequence of itsprotein product. The boundaries of the coding sequence are generallydetermined by an open reading frame, which usually begins with the ATGstart codon or alternative start codons such as GTG and TTG. The codingsequence may a DNA, cDNA, or recombinant nucleotide sequence

Expression Vector

The term “expression” includes any step involved in the production ofthe polypeptide including, but not limited to, transcription,post-transcriptional modification, translation, post-translationalmodification, and secretion.

The term “expression vector” is defined herein as a linear or circularDNA molecule that comprises a polynucleotide encoding a polypeptide ofthe invention, and which is operably linked to additional nucleotidesthat provide for its expression.

A nucleic acid sequence encoding a phytase variant of the invention canbe expressed using an expression vector which typically includes controlsequences encoding a promoter, operator, ribosome binding site,translation initiation signal, and, optionally, a repressor gene orvarious activator genes.

The recombinant expression vector carrying the DNA sequence encoding aphytase variant of the invention may be any vector which mayconveniently be subjected to recombinant DNA procedures, and the choiceof vector will often depend on the host cell into which it is to beintroduced. The vector may be one which, when introduced into a hostcell, is integrated into the host cell genome and replicated togetherwith the chromosome(s) into which it has been integrated.

The phytase variant may also be co-expressed together with at least oneother enzyme of animal feed interest, such as a phytase, phosphatase,xylanase, galactanase, alpha-galactosidase, protease, phospholipase,amylase, and/or beta-glucanase. The enzymes may be co-expressed fromdifferent vectors, from one vector, or using a mixture of bothtechniques. When using different vectors, the vectors may have differentselectable markers, and different origins of replication. When usingonly one vector, the genes can be expressed from one or more promoters.If cloned under the regulation of one promoter (di- or multi-cistronic),the order in which the genes are cloned may affect the expression levelsof the proteins. The phytase variant may also be expressed as a fusionprotein, i.e., that the gene encoding the phytase variant has been fusedin frame to the gene encoding another protein. This protein may beanother enzyme or a functional domain from another enzyme.

Host Cells

The term “host cell”, as used herein, includes any cell type which issusceptible to transformation, transfection, transduction, and the likewith a nucleic acid construct comprising a polynucleotide of the presentinvention.

The present invention also relates to recombinant host cells, comprisinga polynucleotide of the present invention, which are advantageously usedin the recombinant production of the polypeptides. A vector comprising apolynucleotide of the present invention is introduced into a host cellso that the vector is maintained as a chromosomal integrant or as aself-replicating extra-chromosomal vector as described earlier. The term“host cell” encompasses any progeny of a parent cell that is notidentical to the parent cell due to mutations that occur duringreplication. The choice of a host cell will to a large extent dependupon the gene encoding the polypeptide and its source.

The host cell may be a unicellular microorganism, e.g., a prokaryote, ora non-unicellular microorganism, e.g., a eukaryote.

Useful unicellular microorganisms are bacterial cells such as grampositive bacteria including, but not limited to, a Bacillus cell, e.g.,Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus brevis,Bacillus circulans, Bacillus clausii, Bacillus coagulans, Bacilluslautus, Bacillus lentus, Bacillus licheniformis, Bacillus megaterium,Bacillus stearothermophilus, Bacillus subtilis, and Bacillusthuringiensis; or a Streptomyces cell, e.g., Streptomyces lividans andStreptomyces murinus, or gram negative bacteria such as E. coli andPseudomonas sp. In a preferred aspect, the bacterial host cell is aBacillus lentus, Bacillus licheniformis, Bacillus stearothermophilus, orBacillus subtilis cell. In another preferred aspect, the Bacillus cellis an alkalophilic Bacillus.

The introduction of a vector into a bacterial host cell may, forinstance, be effected by protoplast transformation (see, e.g., Chang andCohen, 1979, Molecular General Genetics 168: 111-115), using competentcells (see, e.g., Young and Spizizin, 1961, Journal of Bacteriology 81:823-829, or Dubnau and Davidoff-Abelson, 1971, Journal of MolecularBiology 56: 209-221), electroporation (see, e.g., Shigekawa and Dower,1988, Biotechniques 6: 742-751), or conjugation (see, e.g., Koehler andThorne, 1987, Journal of Bacteriology 169: 5771-5278).

The host cell may also be a eukaryote, such as a mammalian, insect,plant, or fungal cell.

In a preferred aspect, the host cell is a fungal cell. “Fungi” as usedherein includes the phyla Ascomycota, Basidiomycota, Chytridiomycota,and Zygomycota (as defined by Hawksworth et al., In, Ainsworth andBisby's Dictionary of The Fungi, 8th edition, 1995, CAB International,University Press, Cambridge, UK) as well as the Oomycota (as cited inHawksworth et al., 1995, supra, page 171) and all mitosporic fungi(Hawksworth et al., 1995, supra).

In a more preferred aspect, the fungal host cell is a yeast cell.“Yeast” as used herein includes ascosporogenous yeast (Endomycetales),basidiosporogenous yeast, and yeast belonging to the Fungi Imperfecti(Blastomycetes). Since the classification of yeast may change in thefuture, for the purposes of this invention, yeast shall be defined asdescribed in Biology and Activities of Yeast (Skinner, F. A., Passmore,S. M., and Davenport, R. R., eds, Soc. App. Bacteriol. Symposium SeriesNo. 9, 1980).

In an even more preferred aspect, the yeast host cell is a Candida,Hansenula, Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, orYarrowia cell.

In a most preferred aspect, the yeast host cell is a Pichia pastoris,Pichia methanolica, Saccharomyces carlsbergensis, Saccharomycescerevisiae, Saccharomyces diastaticus, Saccharomyces douglasii,Saccharomyces kluyveri, Saccharomyces norbensis or Saccharomycesoviformis cell. In another most preferred aspect, the yeast host cell isa Kluyveromyces lactis cell. In another most preferred aspect, the yeasthost cell is a Yarrowia lipolytica cell.

In another more preferred aspect, the fungal host cell is a filamentousfungal cell. “Filamentous fungi” include all filamentous forms of thesubdivision Eumycota and Oomycota (as defined by Hawksworth et al.,1995, supra). The filamentous fungi are generally characterized by amycelial wall composed of chitin, cellulose, glucan, chitosan, mannan,and other complex polysaccharides. Vegetative growth is by hyphalelongation and carbon catabolism is obligately aerobic. In contrast,vegetative growth by yeasts such as Saccharomyces cerevisiae is bybudding of a unicellular thallus and carbon catabolism may befermentative.

In an even more preferred aspect, the filamentous fungal host cell is anAcremonium, Aspergillus, Aureobasidium, Bjerkandera, Ceriporiopsis,Coprinus, Coriolus, Cryptococcus, Filobasidium, Fusarium, Humicola,Magnaporthe, Mucor, Myceliophthora, Neocallimastix, Neurospora,Paecilomyces, Penicillium, Phanerochaete, Phiebia, Piromyces, Pleurotus,Schizophyllum, Talaromyces, Thermoascus, Thielavia, Tolypocladium,Trametes, or Trichoderma cell.

In a most preferred aspect, the filamentous fungal host cell is anAspergillus awamori, Aspergillus fumigatus, Aspergillus foetidus,Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger orAspergillus oryzae cell. In another most preferred aspect, thefilamentous fungal host cell is a Fusarium bactridioides, Fusariumcerealis, Fusarium crookwellense, Fusarium culmorum, Fusariumgraminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi,Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusariumsambucinum, Fusarium sarcochroum, Fusarium sporotrichioides, Fusariumsuiphureum, Fusarium torulosum, Fusarium trichothecioides, or Fusariumvenenatum cell. In another most preferred aspect, the filamentous fungalhost cell is a Bjerkandera adusta, Ceriporiopsis aneirina, Ceriporiopsisaneirina, Ceriporiopsis caregiea, Ceriporiopsis gilvescens,Ceriporiopsis pannocinta, Ceriporiopsis rivulosa, Ceriporiopsis subrufa,Ceriporiopsis subvermispora, Coprinus cinereus, Coriolus hirsutus,Humicola insolens, Humicola lanuginosa, Mucor miehei, Myceliophthorathermophila, Neurospora crassa, Penicillium purpurogenum, Phanerochaetechrysosporium, Phiebia radiata, Pleurotus eryngii, Thielavia terrestris,Trametes villosa, Trametes versicolor, Trichoderma harzianum,Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma reesei,or Trichoderma viride strain cell.

Fungal cells may be transformed by a process involving protoplastformation, transformation of the protoplasts, and regeneration of thecell wall in a manner known per se. Suitable procedures fortransformation of Aspergillus and Trichoderma host cells are describedin EP 238 023 and Yelton et al., 1984, Proceedings of the NationalAcademy of Sciences USA 81: 1470-1474. Suitable methods for transformingFusarium species are described by Malardier et al., 1989, Gene 78:147-156, and WO 96/00787. Yeast may be transformed using the proceduresdescribed by Becker and Guarente, In Abelson, J. N. and Simon, M. I.,editors, Guide to Yeast Genetics and Molecular Biology, Methods inEnzymology, Volume 194, pp 182-187, Academic Press, Inc., New York; Itoet al., 1983, Journal of Bacteriology 153: 163; and Hinnen et al., 1978,Proceedings of the National Academy of Sciences USA 75: 1920.

Methods of Production

The present invention also relates to methods for producing a phytase ofthe present invention comprising (a) cultivating a host cell underconditions conducive for production of the phytase; and (b) recoveringthe phytase.

In the production methods of the present invention, the cells arecultivated in a nutrient medium suitable for production of thepolypeptide using methods well known in the art. For example, the cellmay be cultivated by shake flask cultivation, and small-scale orlarge-scale fermentation (including continuous, batch, fed-batch, orsolid state fermentations) in laboratory or industrial fermentorsperformed in a suitable medium and under conditions allowing thepolypeptide to be expressed and/or isolated. The cultivation takes placein a suitable nutrient medium comprising carbon and nitrogen sources andinorganic salts, using procedures known in the art. Suitable media areavailable from commercial suppliers or may be prepared according topublished compositions (e.g., in catalogues of the American Type CultureCollection). If the polypeptide is secreted into the nutrient medium,the polypeptide can be recovered directly from the medium. If thepolypeptide is not secreted, it can be recovered from cell lysates.

The resulting polypeptide may be recovered using methods known in theart. For example, the polypeptide may be recovered from the nutrientmedium by conventional procedures including, but not limited to,centrifugation, filtration, extraction, spray-drying, evaporation, orprecipitation.

The polypeptides of the present invention may be purified by a varietyof procedures known in the art including, but not limited to,chromatography (e.g., ion exchange, affinity, hydrophobic,chromatofocusing, and size exclusion), electrophoretic procedures (e.g.,preparative isoelectric focusing), differential solubility (e.g.,ammonium sulfate precipitation), SDS-PAGE, or extraction (see, e.g.,Protein Purification, J.-C. Janson and Lars Ryden, editors, VCHPublishers, New York, 1989).

Transgenic Plants

The present invention also relates to a transgenic plant, plant part, orplant cell which has been transformed with a nucleotide sequenceencoding a polypeptide having phytase activity of the present inventionso as to express and produce the polypeptide in recoverable quantities.The polypeptide may be recovered from the plant or plant part.Alternatively, the plant or plant part containing the recombinantpolypeptide may be used as such for improving the quality of a food orfeed, e.g., improving nutritional value, palatability, and rheologicalproperties, or to destroy an antinutritive factor.

In a particular embodiment, the polypeptide is targeted to the endospermstorage vacuoles in seeds. This can be obtained by synthesizing it as aprecursor with a suitable signal peptide, see Horvath et al., 2000, PNAS97(4): 1914-1919.

The transgenic plant can be dicotyledonous (a dicot) or monocotyledonous(a monocot) or engineered variants thereof. Examples of monocot plantsare grasses, such as meadow grass (blue grass, Poa), forage grass suchas Festuca, Lolium, temperate grass, such as Agrostis, and cereals,e.g., wheat, oats, rye, barley, rice, sorghum, triticale (stabilizedhybrid of wheat (Triticum) and rye (Secale), and maize (corn). Examplesof dicot plants are tobacco, legumes, such as sunflower (Helianthus),cotton (Gossypium), lupins, potato, sugar beet, pea, bean and soybean,and cruciferous plants (family Brassicaceae), such as cauliflower, rapeseed, and the closely related model organism Arabidopsis thaliana.Low-phytate plants as described in, e.g., U.S. Pat. Nos. 5,689,054 and6,111,168 are examples of engineered plants.

Examples of plant parts are stem, callus, leaves, root, fruits, seeds,and tubers, as well as the individual tissues comprising these parts,e.g., epidermis, mesophyll, parenchyma, vascular tissues, meristems.Also specific plant cell compartments, such as chloroplast, apoplast,mitochondria, vacuole, peroxisomes, and cytoplasm are considered to be aplant part. Furthermore, any plant cell, whatever the tissue origin, isconsidered to be a plant part. Likewise, plant parts such as specifictissues and cells isolated to facilitate the utilisation of theinvention are also considered plant parts, e.g., embryos, endosperms,aleurone and seed coats.

Also included within the scope of the present invention are the progenyof such plants, plant parts and plant cells.

The transgenic plant or plant cell expressing a polypeptide of thepresent invention may be constructed in accordance with methods known inthe art. Briefly, the plant or plant cell is constructed byincorporating one or more expression constructs encoding a polypeptideof the present invention into the plant host genome and propagating theresulting modified plant or plant cell into a transgenic plant or plantcell.

Conveniently, the expression construct is a nucleic acid construct whichcomprises a nucleic acid sequence encoding a polypeptide of the presentinvention operably linked with appropriate regulatory sequences requiredfor expression of the nucleic acid sequence in the plant or plant partof choice. Furthermore, the expression construct may comprise aselectable marker useful for identifying host cells into which theexpression construct has been integrated and DNA sequences necessary forintroduction of the construct into the plant in question (the latterdepends on the DNA introduction method to be used).

The choice of regulatory sequences, such as promoter and terminatorsequences and optionally signal or transit sequences are determined, forexample, on the basis of when, where, and how the polypeptide is desiredto be expressed. For instance, the expression of the gene encoding apolypeptide of the present invention may be constitutive or inducible,or may be developmental, stage or tissue specific, and the gene productmay be targeted to a specific cell compartment, tissue or plant partsuch as seeds or leaves. Regulatory sequences are, for example,described by Tague et al., 1988, Plant Physiology 86: 506.

For constitutive expression, the following promoters may be used: The35S-CaMV promoter (Franck et al., 1980, Cell 21: 285-294), the maizeubiquitin 1 (Christensen A H, Sharrock R A and Quail 1992. Maizepolyubiquitin genes: structure, thermal perturbation of expression andtranscript splicing, and promoter activity following transfer toprotoplasts by electroporation), or the rice actin 1 promoter (PlantMol. Biol. 18: 675-689; Zhang et al., Analysis of rice Act1 5′ regionactivity in transgenic rice plants, Plant Cell 3: 1155-1165).Organ-specific promoters may be, for example, a promoter from storagesink tissues such as seeds, potato tubers, and fruits (Edwards &Coruzzi, 1990, Ann. Rev. Genet. 24: 275-303), or from metabolic sinktissues such as meristems (Ito et al., 1994, Plant Mol. Biol. 24:863-878), a seed specific promoter such as the glutelin, prolamin,globulin, or albumin promoter from rice (Wu et al., 1998, Plant and CellPhysiology 39: 885-889), a Vicia faba promoter from the legumin B4 andthe unknown seed protein gene from Vicia faba (Conrad et al., 1998,Journal of Plant Physiology 152: 708-711), a promoter from a seed oilbody protein (Chen et al., 1998, Plant and Cell Physiology 39: 935-941),the storage protein napA promoter from Brassica napus, or any other seedspecific promoter known in the art, e.g., as described in WO 91/14772.Furthermore, the promoter may be a leaf specific promoter such as therbcs promoter from rice or tomato (Kyozuka et al., 1993, PlantPhysiology 102: 991-1000, the chlorella virus adenine methyltransferasegene promoter (Mitra and Higgins, 1994, Plant Molecular Biology 26:85-93), or the aldP gene promoter from rice (Kagaya et al., 1995,Molecular and General Genetics 248: 668-674), or a wound induciblepromoter such as the potato pin2 promoter (Xu et al., 1993, PlantMolecular Biology 22: 573-588). Likewise, the promoter may be inducibleby abiotic treatments such as temperature, drought or alterations insalinity or inducible by exogenously applied substances that activatethe promoter, e.g., ethanol, oestrogens, plant hormones like ethylene,abscisic acid, gibberellic acid, and/or heavy metals.

A promoter enhancer element may also be used to achieve higherexpression of the polypeptide in the plant. For instance, the promoterenhancer element may be an intron which is placed between the promoterand the nucleotide sequence encoding a polypeptide of the presentinvention. For instance, Xu et al., 1993, supra disclose the use of thefirst intron of the rice actin 1 gene to enhance expression.

Still further, the codon usage may be optimized for the plant species inquestion to improve expression (see Horvath et al. referred to above).

The selectable marker gene and any other parts of the expressionconstruct may be chosen from those available in the art.

The nucleic acid construct is incorporated into the plant genomeaccording to conventional techniques known in the art, includingAgrobacterium-mediated transformation, virus-mediated transformation,microinjection, particle bombardment, biolistic transformation, andelectroporation (Gasser et al., 1990, Science 244: 1293; Potrykus, 1990,Bio/Technology 8: 535; Shimamoto et al., 1989, Nature 338: 274).

Presently, Agrobacterium tumefaciens-mediated gene transfer is themethod of choice for generating transgenic dicots (for a review, seeHooykas and Schilperoort, 1992, Plant Molecular Biology 19: 15-38), andit can also be used for transforming monocots, although othertransformation methods are more often used for these plants. Presently,the method of choice for generating transgenic monocots, supplementingthe Agrobacterium approach, is particle bombardment (microscopic gold ortungsten particles coated with the transforming DNA) of embryonic callior developing embryos (Christou, 1992, Plant Journal 2: 275-281;Shimamoto, 1994, Current Opinion Biotechnology 5: 158-162; Vasil et al.,1992, Bio/Technology 10: 667-674). An alternative method fortransformation of monocots is based on protoplast transformation asdescribed by Omirulleh et al., 1993, Plant Molecular Biology 21:415-428.

Following transformation, the transformants having incorporated thereinthe expression construct are selected and regenerated into whole plantsaccording to methods well-known in the art. Often the transformationprocedure is designed for the selective elimination of selection geneseither during regeneration or in the following generations by using,e.g., co-transformation with two separate T-DNA constructs or sitespecific excision of the selection gene by a specific recombinase.

The present invention also relates to methods for producing apolypeptide of the present invention comprising (a) cultivating atransgenic plant or a plant cell comprising a nucleic acid sequenceencoding a polypeptide having phytase activity of the present inventionunder conditions conducive for production of the polypeptide; and (b)recovering the polypeptide.

Transgenic Animals

The present invention also relates to a transgenic, non-human animal andproducts or elements thereof, examples of which are body fluids such asmilk and blood, organs, flesh, and animal cells. Techniques forexpressing proteins, e.g., in mammalian cells, are known in the art,see, e.g., the handbook Protein Expression: A Practical Approach,Higgins and Hames (eds), Oxford University Press (1999), and the threeother handbooks in this series relating to Gene Transcription, RNAprocessing, and Post-translational Processing. Generally speaking, toprepare a transgenic animal, selected cells of a selected animal aretransformed with a nucleic acid sequence encoding a polypeptide havingphytase activity of the present invention so as to express and producethe polypeptide. The polypeptide may be recovered from the animal, e.g.,from the milk of female animals, or the polypeptide may be expressed tothe benefit of the animal itself, e.g., to assist the animal'sdigestion. Examples of animals are mentioned below in the section headedAnimal Feed.

To produce a transgenic animal with a view to recovering the polypeptidefrom the milk of the animal, a gene encoding the polypeptide may beinserted into the fertilized eggs of an animal in question, e.g., by useof a transgene expression vector which comprises a suitable milk proteinpromoter, and the gene encoding the polypeptide. The transgeneexpression vector is microinjected into fertilized eggs, and preferablypermanently integrated into the chromosome. Once the egg begins to growand divide, the potential embryo is implanted into a surrogate mother,and animals carrying the transgene are identified. The resulting animalcan then be multiplied by conventional breeding. The polypeptide may bepurified from the animal's milk, see, e.g., Meade, H. M. et al. (1999):Expression of recombinant proteins in the milk of transgenic animals,Gene expression systems: Using nature for the art of expression. J. M.Fernandez and J. P. Hoeffler (eds.), Academic Press.

In the alternative, in order to produce a transgenic non-human animalthat carries in the genome of its somatic and/or germ cells a nucleicacid sequence including a heterologous transgene construct including atransgene encoding the polypeptide, the transgene may be operably linkedto a first regulatory sequence for salivary gland specific expression ofthe polypeptide, as disclosed in WO 00/64247.

Compositions and Uses

In still further aspects, the present invention relates to compositionscomprising a polypeptide of the present invention, as well as methods ofusing these.

The polypeptide compositions may be prepared in accordance with methodsknown in the art and may be in the form of a liquid or a drycomposition. For instance, the polypeptide composition may be in theform of granulates or microgranulates. The polypeptide to be included inthe composition may be stabilized in accordance with methods known inthe art.

The phytase of the invention can be used for degradation, in anyindustrial context, of, for example, phytate, phytic acid, and/or themono-, di-, tri-, tetra- and/or penta-phosphates of myo-inositol. It iswell known that the phosphate moieties of these compounds chelatesdivalent and trivalent cations such as metal ions, i.a. thenutritionally essential ions of calcium, iron, zinc and magnesium aswell as the trace minerals manganese, copper and molybdenum. Besides,the phytic acid also to a certain extent binds proteins by electrostaticinteraction.

Accordingly, preferred uses of the polypeptides of the invention are inanimal feed preparations (including human food) or in additives for suchpreparations.

In a particular embodiment, the polypeptide of the invention can be usedfor improving the nutritional value of an animal feed. Non-limitingexamples of improving the nutritional value of animal feed (includinghuman food), are: Improving feed digestibility; promoting growth of theanimal; improving feed utilization; improving bio-availability ofproteins; increasing the level of digestible phosphate; improving therelease and/or degradation of phytate; improving bio-availability oftrace minerals; improving bio-availability of macro minerals;eliminating the need for adding supplemental phosphate, trace minerals,and/or macro minerals; and/or improving egg shell quality. Thenutritional value of the feed is therefore increased, and the growthrate and/or weight gain and/or feed conversion (i.e., the weight ofingested feed relative to weight gain) of the animal may be improved.

Furthermore, the polypeptide of the invention can be used for reducingphytate level of manure.

Animals, Animal Feed, and Animal Feed Additives

The term animal includes all animals, including human beings. Examplesof animals are non-ruminants, and ruminants. Ruminant animals include,for example, animals such as sheep, goat, and cattle, e.g. cow such asbeef cattle and dairy cows. In a particular embodiment, the animal is anon-ruminant animal. Non-ruminant animals include mono-gastric animals,e.g., pig or swine (including, but not limited to, piglets, growingpigs, and sows); poultry such as turkeys, ducks and chickens (includingbut not limited to broiler chicks, layers); fish (including but notlimited to salmon, trout, tilapia, catfish and carp); and crustaceans(including but not limited to shrimp and prawn).

The term feed or feed composition means any compound, preparation,mixture, or composition suitable for, or intended for intake by ananimal.

In the use according to the invention the polypeptide can be fed to theanimal before, after, or simultaneously with the diet. The latter ispreferred.

In a particular embodiment, the polypeptide, in the form in which it isadded to the feed, or when being included in a feed additive, issubstantially pure. In a particular embodiment it is well-defined. Theterm “well-defined” means that the phytase preparation is at least 50%pure as determined by Size-exclusion chromatography (see Example 12 ofWO 01/58275). In other particular embodiments the phytase preparation isat least 60, 70, 80, 85, 88, 90, 92, 94, or at least 95% pure asdetermined by this method.

A substantially pure, and/or well-defined polypeptide preparation isadvantageous. For instance, it is much easier to dose correctly to thefeed a polypeptide that is essentially free from interfering orcontaminating other polypeptides. The term dose correctly refers inparticular to the objective of obtaining consistent and constantresults, and the capability of optimising dosage based upon the desiredeffect.

For the use in animal feed, however, the phytase polypeptide of theinvention need not be that pure; it may, e.g., include otherpolypeptides, in which case it could be termed a phytase preparation.

The phytase preparation can be (a) added directly to the feed (or useddirectly in a treatment process of proteins), or (b) it can be used inthe production of one or more intermediate compositions such as feedadditives or premixes that is subsequently added to the feed (or used ina treatment process). The degree of purity described above refers to thepurity of the original polypeptide preparation, whether used accordingto (a) or (b) above.

Polypeptide preparations with purities of this order of magnitude are inparticular obtainable using recombinant methods of production, whereasthey are not so easily obtained and also subject to a much higherbatch-to-batch variation when the polypeptide is produced by traditionalfermentation methods.

Such polypeptide preparation may of course be mixed with otherpolypeptides.

The polypeptide can be added to the feed in any form, be it as arelatively pure polypeptide, or in admixture with other componentsintended for addition to animal feed, i.e., in the form of animal feedadditives, such as the so-called pre-mixes for animal feed.

In a further aspect the present invention relates to compositions foruse in animal feed, such as animal feed, and animal feed additives,e.g., premixes.

Apart from the polypeptide of the invention, the animal feed additivesof the invention contain at least one fat-soluble vitamin, and/or atleast one water soluble vitamin, and/or at least one trace mineral. Thefeed additive may also contain at least one macro mineral.

Further, optional, feed-additive ingredients are colouring agents, e.g.,carotenoids such as beta-carotene, astaxanthin, and lutein; aromacompounds; stabilisers; antimicrobial peptides; polyunsaturated fattyacids; reactive oxygen generating species; and/or at least one otherpolypeptide selected from amongst phytase (EC 3.1.3.8 or 3.1.3.26);phosphatase (EC 3.1.3.1; EC 3.1.3.2; EC 3.1.3.39); xylanase (EC3.2.1.8); galactanase (EC 3.2.1.89); alpha-galactosidase (EC 3.2.1.22);protease (EC 3.4.-.-), phospholipase A1 (EC 3.1.1.32); phospholipase A2(EC 3.1.1.4); lysophospholipase (EC 3.1.1.5); phospholipase C (3.1.4.3);phospholipase D (EC 3.1.4.4); amylase such as, for example,alpha-amylase (EC 3.2.1.1); and/or beta-glucanase (EC 3.2.1.4 or EC3.2.1.6).

In a particular embodiment these other polypeptides are well-defined (asdefined above for phytase preparations).

The phytase of the invention may also be combined with other phytases,for example ascomycete phytases such as Aspergillus phytases, forexample derived from Aspergillus ficuum, Aspergillus niger, orAspergillus awamori; or basidiomycete phytases, for example derived fromPeniophora lycii, Agrocybe pediades, Trametes pubescens, or Paxillusinvolutus; or derivatives, fragments or variants thereof which havephytase activity.

Thus, in preferred embodiments of the use in animal feed of theinvention, and in preferred embodiments of the animal feed additive andthe animal feed of the invention, the phytase of the invention iscombined with such phytases.

Examples of antimicrobial peptides (AMP's) are CAP18, Leucocin A,Tritrpticin, Protegrin-1, Thanatin, Defensin, Lactoferrin,Lactoferricin, and Ovispirin such as Novispirin (Robert Lehrer, 2000),Plectasins, and Statins, including the compounds and polypeptidesdisclosed in WO 03/044049 and WO 03/048148, as well as variants orfragments of the above that retain antimicrobial activity.

Examples of antifungal polypeptides (AFP's) are the Aspergillusgiganteus, and Aspergillus niger peptides, as well as variants andfragments thereof which retain antifungal activity, as disclosed in WO94/01459 and WO 02/90384.

Examples of polyunsaturated fatty acids are C18, C20 and C22polyunsaturated fatty acids, such as arachidonic acid, docosohexaenoicacid, eicosapentaenoic acid and gamma-linoleic acid.

Examples of reactive oxygen generating species are chemicals such asperborate, persulphate, or percarbonate; and polypeptides such as anoxidase, an oxygenase or a syntethase.

Usually fat- and water-soluble vitamins, as well as trace minerals formpart of a so-called premix intended for addition to the feed, whereasmacro minerals are usually separately added to the feed. Either of thesecomposition types, when enriched with a polypeptide of the invention, isan animal feed additive of the invention.

In a particular embodiment, the animal feed additive of the invention isintended for being included (or prescribed as having to be included) inanimal diets or feed at levels of 0.01 to 10.0%; more particularly 0.05to 5.0%; or 0.2 to 1.0% (% meaning g additive per 100 g feed). This isso in particular for premixes.

The following are non-exclusive lists of examples of these components:

Examples of fat-soluble vitamins are vitamin A, vitamin D3, vitamin E,and vitamin K, e.g., vitamin K3.

Examples of water-soluble vitamins are vitamin B12, biotin and choline,vitamin B1, vitamin B2, vitamin B6, niacin, folic acid andpanthothenate, e.g., Ca-D-panthothenate.

Examples of trace minerals are manganese, zinc, iron, copper, iodine,selenium, and cobalt.

Examples of macro minerals are calcium, phosphorus and sodium.

The nutritional requirements of these components (exemplified withpoultry and piglets/pigs) are listed in Table A of WO 01/58275.Nutritional requirement means that these components should be providedin the diet in the concentrations indicated.

In the alternative, the animal feed additive of the invention comprisesat least one of the individual components specified in Table A of WO01/58275. At least one means either of, one or more of, one, or two, orthree, or four and so forth up to all thirteen, or up to all fifteenindividual components. More specifically, this at least one individualcomponent is included in the additive of the invention in such an amountas to provide an in-feed-concentration within the range indicated incolumn four, or column five, or column six of Table A.

The present invention also relates to animal feed compositions. Animalfeed compositions or diets have a relatively high content of protein.Poultry and pig diets can be characterised as indicated in Table B of WO01/58275, columns 2-3. Fish diets can be characterised as indicated incolumn 4 of this Table B. Furthermore such fish diets usually have acrude fat content of 200-310 g/kg. WO 01/58275 corresponds to U.S.application Ser. No. 09/779,334 which is hereby incorporated byreference.

An animal feed composition according to the invention has a crudeprotein content of 50-800 g/kg, and furthermore comprises at least onepolypeptide as claimed herein.

Furthermore, or in the alternative (to the crude protein contentindicated above), the animal feed composition of the invention has acontent of metabolisable energy of 10-30 MJ/kg; and/or a content ofcalcium of 0.1-200 g/kg; and/or a content of available phosphorus of0.1-200 g/kg; and/or a content of methionine of 0.1-100 g/kg; and/or acontent of methionine plus cysteine of 0.1-150 g/kg; and/or a content oflysine of 0.5-50 g/kg.

In particular embodiments, the content of metabolisable energy, crudeprotein, calcium, phosphorus, methionine, methionine plus cysteine,and/or lysine is within any one of ranges 2, 3, 4 or 5 in Table B of WO01/58275 (R. 2-5).

Crude protein is calculated as nitrogen (N) multiplied by a factor 6.25,i.e., Crude protein (g/kg)=N (g/kg)×6.25. The nitrogen content isdetermined by the Kjeldahl method (A.O.A.C., 1984, Official Methods ofAnalysis 14th ed., Association of Official Analytical Chemists,Washington D.C.).

Metabolisable energy can be calculated on the basis of the NRCpublication Nutrient requirements in swine, ninth revised edition 1988,subcommittee on swine nutrition, committee on animal nutrition, board ofagriculture, national research council. National Academy Press,Washington, D.C., pp. 2-6, and the European Table of Energy Values forPoultry Feed-stuffs, Spelderholt centre for poultry research andextension, 7361 DA Beekbergen, The Netherlands. Grafisch bedrijf Ponsen& Iooijen by, Wageningen. ISBN 90-71463-12-5.

The dietary content of calcium, available phosphorus and amino acids incomplete animal diets is calculated on the basis of feed tables such asVeevoedertabel 1997, gegevens over chemische samenstelling,verteerbaarheid en voederwaarde van voedermiddelen, CentralVeevoederbureau, Runderweg 6, 8219 pk Lelystad. ISBN 90-72839-13-7.

In a particular embodiment, the animal feed composition of the inventioncontains at least one protein. The protein may be an animal protein,such as meat and bone meal, and/or fish meal; or it may be a vegetableprotein. The term vegetable proteins as used herein refers to anycompound, composition, preparation or mixture that includes at least oneprotein derived from or originating from a vegetable, including modifiedproteins and protein-derivatives. In particular embodiments, the proteincontent of the vegetable proteins is at least 10, 20, 30, 40, 50, or 60%(w/w).

Vegetable proteins may be derived from vegetable protein sources, suchas legumes and cereals, for example materials from plants of thefamilies Fabaceae (Leguminosae), Cruciferaceae, Chenopodiaceae, andPoaceae, such as soy bean meal, lupin meal and rapeseed meal.

In a particular embodiment, the vegetable protein source is materialfrom one or more plants of the family Fabaceae, e.g., soybean, lupine,pea, or bean.

In another particular embodiment, the vegetable protein source ismaterial from one or more plants of the family Chenopodiaceae, e.g.,beet, sugar beet, spinach or quinoa.

Other examples of vegetable protein sources are rapeseed, sunflowerseed, cotton seed, and cabbage.

Soybean is a preferred vegetable protein source.

Other examples of vegetable protein sources are cereals such as barley,wheat, rye, oat, maize (corn), rice, triticale, and sorghum.

In still further particular embodiments, the animal feed composition ofthe invention contains 0-80% maize; and/or 0-80% sorghum; and/or 0-70%wheat; and/or 0-70% Barley; and/or 0-30% oats; and/or 0-40% soybeanmeal; and/or 0-25% fish meal; and/or 0-25% meat and bone meal; and/or0-20% whey.

Animal diets can, e.g., be manufactured as mash feed (non pelleted) orpelleted feed. Typically, the milled feed-stuffs are mixed andsufficient amounts of essential vitamins and minerals are addedaccording to the specifications for the species in question.Polypeptides can be added as solid or liquid polypeptide formulations.For example, a solid polypeptide formulation is typically added beforeor during the mixing step; and a liquid polypeptide preparation istypically added after the pelleting step. The polypeptide may also beincorporated in a feed additive or premix.

The final polypeptide concentration in the diet is within the range of0.01-200 mg polypeptide protein per kg diet, for example in the range of5-30 mg polypeptide protein per kg animal diet.

The phytase of the invention should of course be applied in an effectiveamount, i.e., in an amount adequate for improving solubilisation and/orimproving nutritional value of feed. It is at present contemplated thatthe polypeptide is administered in one or more of the following amounts(dosage ranges): 0.01-200; 0.01-100; 0.5-100; 1-50; 5-100; 10-100;0.05-50; or 0.10-10—all these ranges being in mg phytase polypeptideprotein per kg feed (ppm).

For determining mg phytase polypeptide protein per kg feed, the phytaseis purified from the feed composition, and the specific activity of thepurified phytase is determined using a relevant assay. The phytaseactivity of the feed composition as such is also determined using thesame assay, and on the basis of these two determinations, the dosage inmg phytase protein per kg feed is calculated.

The same principles apply for determining mg phytase polypeptide proteinin feed additives. Of course, if a sample is available of the phytaseused for preparing the feed additive or the feed, the specific activityis determined from this sample (no need to purify the phytase from thefeed composition or the additive).

Particular Embodiments

The invention also relates to the following particular embodiments:

I. A phytase which has at least 74% identity to SEQ ID NO: 2 and whichcomprises at least one alteration as compared to SEQ ID NO: 2 in atleast one position selected from the following: 1, 2, 3, 4, 5, 31, 41,46, 52, 53, 55, 57, 59, 74, 76, 82, 84, 91, 99, 100, 104, 105, 107, 109,111, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 136, 137,141, 154, 161, 162, 164, 167, 171, 176, 177, 179, 180, 181, 182, 183,184, 185, 186, 196, 199, 200, 202, 203, 218, 223, 239, 240, 241, 247,273, 276, 281, 282, 283, 284, 285, 286, 289, 294, 299, 308, 314, 316,324, 331, 339, 351, 355, 362, 379, 385, 406, 409, 410, and 411;

preferably in at least one position selected from the following: 1, 2,3, 4, 5, 31, 46, 52, 53, 55, 57, 59, 76, 82, 99, 100, 107, 109, 111,114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 137, 141, 161,162, 164, 167, 179, 180, 181, 182, 183, 184, 185, 186, 196, 199, 200,202, 218, 223, 241, 273, 276, 285, 286, 299, 314, 331, 339, 362, 379,385, 406, 410, and 411;

with the proviso that the phytase is not SEQ ID NO: 3, not SEQ ID NO: 4,and not SEQ ID NO: 6.

II. A phytase which has at least 74% identity to SEQ ID NO: 2 and whichcomprises at least one alteration as compared to SEQ ID NO: 2 in atleast one position selected from the following: 1, 2, 3, 4, 5, 41, 46,52, 53, 55, 57, 59, 74, 76, 82, 84, 91, 99, 100, 104, 105, 107, 109,111, 114, 115, 116, 117, 118, 119, 120, 122, 123, 124, 136, 137, 141,154, 161, 162, 164, 167, 171, 176, 177, 179, 180, 181, 182, 183, 184,185, 186, 196, 199, 200, 202, 203, 218, 223, 239, 240, 241, 247, 273,276, 281, 282, 283, 284, 285, 286, 289, 294, 299, 308, 314, 324, 339,351, 355, 362, 379, 385, 406, 409, 410, and 411;

preferably in at least one position selected from the following: 1, 2,3, 4, 5, 46, 52, 53, 55, 57, 59, 76, 82, 99, 100, 107, 109, 111, 114,115, 116, 117, 118, 119, 120, 122, 123, 124, 137, 141, 161, 162, 164,167, 179, 180, 181, 182, 183, 184, 185, 186, 196, 199, 200, 202, 218,223, 241, 273, 276, 285, 286, 299, 314, 339, 362, 379, 385, 406, 410,and 411.

III. A phytase which has at least 74% identity to SEQ ID NO: 2 and whichcomprises at least one alteration as compared to SEQ ID NO: 2 in atleast one position selected from the following: 1, 2, 3, 4, 5, 31, 41,46, 52, 53, 55, 57, 59, 74, 76, 82, 84, 91, 99, 100, 104, 105, 107, 109,111, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 136, 137,141, 154, 161, 162, 164, 167, 171, 176, 177, 179, 180, 181, 182, 183,184, 185, 186, 196, 199, 200, 202, 203, 218, 223, 239, 240, 241, 247,273, 276, 281, 282, 283, 284, 285, 286, 289, 294, 299, 308, 314, 316,324, 331, 339, 351, 355, 362, 379, 385, 406, 409, 410, and 411;

preferably in at least one position selected from the followingpreferably in at least one position selected from the following: 1, 2,3, 4, 5, 31, 46, 52, 53, 55, 57, 59, 76, 82, 99, 100, 107, 109, 111,114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 137, 141, 161,162, 164, 167, 179, 180, 181, 182, 183, 184, 185, 186, 196, 199, 200,202, 218, 223, 241, 273, 276, 285, 286, 299, 314, 331, 339, 362, 379,385, 406, 410, and 411;

with the proviso that the phytase is not SEQ ID NO: 3, not SEQ ID NO: 4,not SEQ ID NO: 6, and not SEQ ID NO: 9 and the variants thereof listedin FIG. 1.

IV. A phytase which has at least 74% identity to SEQ ID NO: 2 and whichcomprises at least one alteration as compared to SEQ ID NO: 2 in atleast one position selected from the following: 1, 2, 3, 4, 5, 41, 46,52, 53, 55, 57, 59, 74, 76, 82, 84, 91, 99, 100, 104, 105, 107, 109,111, 114, 115, 116, 117, 118, 119, 120, 122, 123, 124, 136, 137, 141,154, 161, 162, 164, 167, 171, 176, 177, 179, 180, 181, 182, 183, 184,185, 186, 196, 199, 200, 202, 203, 218, 223, 239, 240, 241, 247, 273,276, 281, 282, 283, 284, 285, 286, 289, 294, 299, 308, 314, 324, 339,351, 355, 362, 379, 385, 406, 409, 410, and 411

preferably in at least one position selected from the following: 1, 2,3, 4, 5, 46, 52, 53, 55, 57, 59, 76, 82, 99, 100, 107, 109, 111, 114,115, 116, 117, 118, 119, 120, 122, 123, 124, 137, 141, 161, 162, 164,167, 179, 180, 181, 182, 183, 184, 185, 186, 196, 199, 200, 202, 218,223, 241, 273, 276, 285, 286, 299, 314, 339, 362, 379, 385, 406, 410,and 411;

with the proviso that the phytase is not SEQ ID NO: 9 and the variantsthereof listed in FIG. 1.

V. A phytase which has at least 74% identity to SEQ ID NO: 2 and whichcomprises at least one alteration as compared to SEQ ID NO: 2 in atleast one position selected from the following: 4, 5, 41, 46, 59, 82,84, 91, 99, 105, 107, 109, 111, 115, 116, 117, 119, 122, 123, 124, 136,137, 141, 161, 162, 164, 167, 171, 176, 179, 180, 186, 196, 199, 200,218, 223, 239, 240, 241, 247, 273, 276, 281, 282, 283, 284, 289, 294,299, 308, 314, 324, 339, 351, 355, 379, 385, 406, 409, 410, and 411;

preferably in at least one position selected from the following: 4, 5,46, 59, 82, 99, 107, 109, 111, 115, 116, 117, 119, 122, 123, 124, 137,141, 161, 162, 164, 167, 179, 180, 186, 196, 199, 200, 218, 223, 241,273, 276, 299, 314, 339, 379, 385, 406, 410, and 411.

VI. A phytase which has at least 74% identity to SEQ ID NO: 2 and whichcomprises at least one of the following alterations: 1*, 2*, 3*, 4P, 5P,31C,T, 41P, 46C,D,E, 52C,E, 53V,Q, 55D,I, 57Y, 59C, 74A, 76G, 82E, 84Y,91C,P, 99C, 100C, 104A, 105F, 107D,E,G, 109A,G, 111P, 114H,N,T, 115Q,116A,E,P,T,Q, 117D,E,K 118I,L,M,T, 119G,K,R,S, 120K,S,T,Q,121A,D,M,P,T,V, 122D, 123P,S, 124L,T,V, 136P, 137P, 141C, 154P, 161P,162C, 164D,E, 167Q, 171T, 176C, 177C, 179G,I,K,N,Q, 180A,E,G,T,181D,G,I,K, 182H,K,S,Q, 183A,L,P,S,V,Q, 184*, 185*, 186*, 196Q, 199C,200K,R, 202N, 203T, 218Q, 223E, 239Q, 240P, 241Q, 247C, 273L,Q, 276K,R,281H, 282P, 283P, 284P, 285G,N,R, 286K,Q, 289P, 294T, 299L, 308A,314G,N, 316D, 324N, 331K, 339D, 351Y, 355P, 362K,R, 379K,R, 385D, 406A,409D,E, 410D,E, and/or 411R,K; and/or wherein the amino acids inposition 179, 180, 181, 182, 183, 184, 185, and 186 have been replacedby QADKP (SEQ ID NO: 17), GEDKP (SEQ ID NO: 18), NGISA (SEQ ID NO: 19),IAGKS (SEQ ID NO: 20), KEKHQ (SEQ ID NO: 21), KEKQQ (SEQ ID NO: 22),KEKKV (SEQ ID NO: 23), or KTDKL (SEQ ID NO: 24);

preferably at least one of the following alterations: 1*, 2*, 3*, 4P,5P, 31C, 46E, 52C,E, 53V, 55D, 57Y, 59C, 76G, 82E, 99C, 100C, 107D,E,G,109A, 111P, 114T, 115Q, 116AT, 117D, 118T, 119K,R,S, 120S, 121D,P,T,122D, 123P, 124L, 137P, 141C, 161P, 162C, 164E, 167Q, 179K, 180E,T,181D,K, 182H,K,Q, 183L,V,Q, 184*, 185*, 186*, 196Q, 199C, 200K, 202N,218Q, 223E, 241Q, 273L, 276K,R, 285G,R, 286Q, 299L, 314G,N, 331K, 339D,362K,R, 379K,R, 385D, 406A, 410D,E, and/or 411R,K; and/or wherein theamino acids in position 179, 180, 181, 182, 183, 184, 185, and 186 havebeen replaced by KEKHQ (SEQ ID NO: 21), KEKQQ (SEQ ID NO: 22), KEKKV(SEQ ID NO: 23), or KTDKL (SEQ ID NO: 24);

with the proviso that the phytase is not SEQ ID NO: 3, not SEQ ID NO: 4,and not SEQ ID NO: 6.

VII. A phytase which has at least 74% identity to SEQ ID NO: 2 and whichcomprises at least one of the following alterations: 1*, 2*, 3*, 4P, 5P,31C,T, 41P, 46C,D,E, 52C,E, 53V,Q, 55I,D, 57Y, 59C, 74A, 76G, 82E, 84Y,91C,P, 99C, 100C, 104A, 105F, 107D,E,G, 109A,G, 111P, 114H,N,T, 115Q,116A,E,P,T,Q 117D,E,K, 118I,M,L,T, 119G,K,R,S, 120K,S,T,Q, 121A,D,M,P,V,122D, 123P,S, 124L,T,V, 136P, 137P, 141C, 154P, 161P, 162C, 164D,E,167Q, 171T, 176C, 177C, 179G,I,K,N,Q, 180A,E,G,T, 181D,G,I,K,182H,K,S,Q, 183A,L,P,S,V,Q, 184*, 185*, 186*, 196Q, 199C, 200K,R, 202N,203T, 218Q, 223E, 239Q, 240P, 241Q, 247C, 273L,Q, 276K,R, 281H, 282P,283P, 284P, 285G,N,R, 286K,Q, 289P, 294T, 299L, 308A, 314G,N, 316D,324N, 339D, 351Y, 355P, 362K,R, 379K,R, 385D, 406A, 409D,E, 410D,E,and/or 411K,R; and/or wherein the amino acids in position 179, 180, 181,182, 183, 184, 185, and 186 have been replaced by QADKP (SEQ ID NO: 17),GEDKP (SEQ ID NO: 18), NGISA (SEQ ID NO: 19), IAGKS (SEQ ID NO: 20),KEKHQ (SEQ ID NO: 21), KEKQQ (SEQ ID NO: 22), KEKKV (SEQ ID NO: 23), orKTDKL (SEQ ID NO: 24);

preferably at least one of the following alterations: 1*, 2*, 3*, 4P,5P, 31C, 46E, 52C,E, 53V, 55D, 57Y, 59C, 76G, 82E, 99C, 100C, 107D,E,G,109A, 111P, 114T, 115Q, 116AT, 117D, 118T, 119K,R,S, 120S, 121D,P, 122D,123P, 124L, 137P, 141C, 161P, 162C, 164E, 167Q, 179K, 180E,T, 181D,K,182H,K,Q, 183L,V,Q, 184*, 185*, 186*, 196Q, 199C, 200K 202N, 218Q, 223E,241Q, 273L, 276K,R, 285G,R, 286Q, 299L, 314G,N, 339D, 362K,R, 379K,R,385D, 406A, 410D,E, and/or 411R,K; and/or wherein the amino acids inposition 179, 180, 181, 182, 183, 184, 185, and 186 have been replacedby KEKHQ (SEQ ID NO: 21), KEKQQ (SEQ ID NO: 22), KEKKV (SEQ ID NO: 23),or KTDKL (SEQ ID NO: 24).

IIX. A phytase which has at least 74% identity to SEQ ID NO: 2 and whichcomprises at least one of the following alterations: 1*, 2*, 3*, 4P, 5P,31C,T, 41P, 46C,D,E, 52C,E, 53V,Q, 55D,I, 57Y, 59C, 74A, 76G, 82E, 84Y,91C,P, 99C, 100C, 104A, 105F, 107D,E,G, 109A,G, 111P, 114H,N,T, 115Q,116A,E,P,T,Q, 117D,E,K, 118I,L,M,T, 119G,K,R,S, 120K,S,T,Q,121A,D,M,P,T,V, 122D, 123P,S, 124L,T,V, 136P, 137P, 141C, 154P, 161P,162C, 164D,E, 167Q, 171T, 176C, 177C, 179G,I,K,N,Q, 180A,E,G,T,181D,G,I,K, 182H,K,S,Q, 183A,L,P,S,V,Q, 184*, 185*, 186*, 196Q, 199C,200K,R, 202N, 203T, 218Q, 223E, 239Q, 240P, 241Q, 247C, 273L,Q, 276K,R,281H, 282P, 283P, 284P, 285G,N,R, 286K,Q, 289P, 294T, 299L, 308A,314G,N, 316D, 324N, 331K, 339D, 351Y, 355P, 362K,R, 379K,R, 385D, 406A,409D,E, 410D,E, and/or 411R,K; and/or wherein the amino acids inposition 179, 180, 181, 182, 183, 184, 185, and 186 have been replacedby QADKP (SEQ ID NO: 17), GEDKP (SEQ ID NO: 18), NGISA (SEQ ID NO: 19),IAGKS (SEQ ID NO: 20), KEKHQ (SEQ ID NO: 21), KEKQQ (SEQ ID NO: 22),KEKKV (SEQ ID NO: 23), or KTDKL (SEQ ID NO: 24);

preferably at least one of the following alterations: 1*, 2*, 3*, 4P,5P, 31C, 46E, 52C,E, 53V, 55D, 57Y, 59C, 76G, 82E, 99C, 100C, 107D,E,G,109A, 111P, 114T, 115Q, 116AT, 117D, 118T, 119K,R,S, 120S, 121D,P, 122D,123P, 124L, 137P, 141C, 161P, 162C, 164E, 167Q, 179K, 180E,T, 181D,K,182H,K,Q, 183L,V,Q, 184*, 185*, 186*, 196Q, 199C, 200K 202N, 218Q, 223E,241Q, 273L, 276K,R, 285G,R, 286Q, 299L, 314G,N, 339D, 362K,R, 379K,R,385D, 406A, 410D,E, and/or 411R,K; and/or wherein the amino acids inposition 179, 180, 181, 182, 183, 184, 185, and 186 have been replacedby KEKHQ (SEQ ID NO: 21), KEKQQ (SEQ ID NO: 22), KEKKV (SEQ ID NO: 23),or KTDKL (SEQ ID NO: 24).

with the proviso that the phytase is not SEQ ID NO: 3, not SEQ ID NO: 4,not SEQ ID NO: 6, and not SEQ ID NO: 9 and the variants thereof listedin FIG. 1.

IX. A phytase which has at least 74% identity to SEQ ID NO: 2 and whichcomprises at least one of the following alterations: 1*, 2*, 3*, 4P, 5P,31C,T, 41P, 46C,D,E, 52C,E, 53V,Q, 55I,D, 57Y, 59C, 74A, 76G, 82E, 84Y,91C,P, 99C, 100C, 104A, 105F, 107D,E,G, 109A,G, 111P, 114H,N,T, 115Q,116A,E,P,T,Q 117D,E,K, 118I,M,L,T, 119G,K,R,S, 120K,S,T,Q, 121A,D,M,P,V,122D, 123P,S, 124L,T,V, 136P, 137P, 141C, 154P, 161P, 162C, 164D,E,167Q, 171T, 176C, 177C, 179G,I,K,N,Q, 180A,E,G,T, 181D,G,I,K,182H,K,S,Q, 183A,L,P,S,V,Q, 184*, 185*, 186*, 196Q, 199C, 200K,R, 202N,203T, 218Q, 223E, 239Q, 240P, 241Q, 247C, 273L,Q, 276K,R, 281H, 282P,283P, 284P, 285G,N,R, 286K,Q, 289P, 294T, 299L, 308A, 314G,N, 316D,324N, 339D, 351Y, 355P, 362K,R, 379K,R, 385D, 406A, 409D,E, 410D,E,and/or 411K,R; and/or wherein the amino acids in position 179, 180, 181,182, 183, 184, 185, and 186 have been replaced by QADKP (SEQ ID NO: 17),GEDKP (SEQ ID NO: 18), NGISA (SEQ ID NO: 19), IAGKS (SEQ ID NO: 20),KEKHQ (SEQ ID NO: 21), KEKQQ (SEQ ID NO: 22), KEKKV (SEQ ID NO: 23), orKTDKL (SEQ ID NO: 24);

preferably at least one of the following alterations: 1*, 2*, 3*, 4P,5P, 31C, 46E, 52C,E, 53V, 55D, 57Y, 59C, 76G, 82E, 99C, 100C, 107D,E,G,109A, 111P, 114T, 115Q, 116AT, 117D, 118T, 119K,R,S, 120S, 121D,P, 122D,123P, 124L, 137P, 141C, 161P, 162C, 164E, 167Q, 179K, 180E,T, 181D,K,182H,K,Q, 183L,V,Q, 184*, 185*, 186*, 196Q, 199C, 200K 202N, 218Q, 223E,241Q, 273L, 276K,R, 285G,R, 286Q, 299L, 314G,N, 339D, 362K,R, 379K,R,385D, 406A, 410D,E, and/or 411R,K; and/or wherein the amino acids inposition 179, 180, 181, 182, 183, 184, 185, and 186 have been replacedby KEKHQ (SEQ ID NO: 21), KEKQQ (SEQ ID NO: 22), KEKKV (SEQ ID NO: 23),or KTDKL (SEQ ID NO: 24).

with the proviso that the phytase is not SEQ ID NO: 9 and the variantsthereof listed in FIG. 1.

X. A phytase which has at least 74% identity to SEQ ID NO: 2 and whichcomprises at least one of the following alterations: A phytase which hasat least 74% identity to SEQ ID NO: 2 and which comprises at least oneof the following alterations: 1*, 2*, 3*, 4P, 5P, 31C,T, 41P, 46C,D,E,52C,E, 53Q, 55D, 57Y, 59C, 74A, 82E, 84Y, 91C,P, 99C, 100C, 104A, 105F,107D,E,G, 109A,G, 111P, 114H,T, 115Q, 116A,E,P,T,Q 117D,E,K, 118I,M,L,T,119G,K,R,S, 120K,S,T,Q, 121A,D,M,V, 122D, 123P,S, 124L,T,V, 136P, 137P,141C, 154P, 161P, 162C, 164D,E, 167Q, 171T, 176C, 177C, 179G,I,K,N,Q,180A,E,G,T, 181D,G,K, 182K,S,Q, 183A,L,S,V,Q, 184*, 185*, 186*, 196Q,199C, 200K,R, 202N, 203T, 218Q, 223E, 239Q, 240P, 241Q, 247C, 273L,Q,276K,R, 281H, 282P, 283P, 284P, 285G,N,R, 286K,Q, 289P, 294T, 299L,308A, 314G,N, 316D, 324N, 339D, 351Y, 355P, 362K,R, 379K,R, 385D, 406A,409D,E, 410D,E, and/or 411K,R; and/or wherein the amino acids inposition 179, 180, 181, 182, 183, 184, 185, and 186 have been replacedby QADKP (SEQ ID NO: 17), GEDKP (SEQ ID NO: 18), NGISA (SEQ ID NO: 19),IAGKS (SEQ ID NO: 20), KEKHQ (SEQ ID NO: 21), KEKQQ (SEQ ID NO: 22),KEKKV (SEQ ID NO: 23), or KTDKL (SEQ ID NO: 24);

preferably at least one of the following alterations: 1*, 2*, 3*, 4P,5P, 31C, 46E, 52C,E, 55D, 57Y, 59C, 82E, 99C, 100C, 107D,E,G, 109A,111P, 114T, 115Q, 116AT, 117D, 118T, 119K,R,S, 120S, 121D, 122D, 123P,124L, 137P, 141C, 161P, 162C, 164E, 167Q, 179K, 180E,T, 181D,K, 182K,Q,183L,V,Q, 184*, 185*, 186*, 196Q, 199C, 200K 202N, 218Q, 223E, 241Q,273L, 276K,R, 285G,R, 286Q, 299L, 314G,N, 339D, 362K,R, 379K,R, 385D,406A, 410D,E, and/or 411R,K; and/or wherein the amino acids in position179, 180, 181, 182, 183, 184, 185, and 186 have been replaced by KEKHQ(SEQ ID NO: 21), KEKQQ (SEQ ID NO: 22), KEKKV (SEQ ID NO: 23), or KTDKL(SEQ ID NO: 24).

XI. A phytase which has at least 74% identity to SEQ ID NO: 2 and whichcomprises at least one of the following alterations: 1H,K,R, 60P, 105E,106A,G, 155F, 157F, 173P, 175L, 188P, 205P, 215M, 231P, 254Y, 280P,330D, and/or 371P;

preferably 1K;

with the proviso that the phytase is not SEQ ID NO: 3, not SEQ ID NO: 4,not SEQ ID NO: 6, and not SEQ ID NO: 9 and the variants thereof listedin FIG. 1.

XII. A phytase which has at least 74% identity to SEQ ID NO: 2 and whichcomprises at least one of the following alterations: 1H,R, 60P, 105E,106A,G, 157F, 173P, 175L, 188P, 205P, 215M, 231P, 254Y, 280P.

XIII. A phytase which has at least 74% identity to SEQ ID NO: 2 andwhich comprises at least one of the following alterations: 52C, 141C,162C, 31C, 52C, 99C, 59C, 100C, 141C/199C, 4P, 5P, 111P, 137P, 161P,52E, 57Y, 76G, 107D, 107G, 109A, 1*, 1*/2*, 1*/2*/3*, 121T, 273L, 285G,286Q, 299L, 362K, 331K/55D, 107E, 46E, 82E, 119R, 119K, 164E, 223E,276R, 276K, 362R, 379R, 379K, 385D, 410D, 410E, 411R, 411K, 53V, 121D,167Q, 196Q, 200K, 202N, 218Q, 241Q, 285N, 314N, 314G, 406A,179K/180E/181K/182H/183Q/184*/185*/186*,179K/180E/181K/182Q/183Q/184*/185*/186*,179K/180E/181K/182K/183V/184*/185*/186*,179K/180T/181D/182K/183L/184*/185*/186*,111P/241Q, 1K,114T/115Q/116A/117D/118T/119S/120S/121P/122D/123P/124L,114T/115Q/116T/117D/118T/119S/120S/121P/122D/123P/124L.XIV. The phytase of any of embodiments I-XIII which is a variant of thephytase of SEQ ID NO: 2.XV. The phytase of any of embodiments I-XIII which is a variant of thephytase of SEQ ID NO: 3.XVI. The phytase of any of embodiments I-XIII which is a variant of thephytase of SEQ ID NO: 4.XVII. The phytase of any of embodiments I-XIII which is a variant of thephytase of SEQ ID NO: 6.XVIII. The phytase of any of embodiments I-XIII which is a variant ofthe phytase of SEQ ID NO: 9.XIX. The phytase of any of embodiments I-XIII which is a variant of anyone of the phytase variants related to SEQ ID NO: 9 and listed in FIG.1.XX. The phytase of any of embodiments I-XIX which furthermore comprisesa substitution or a combination of substitutions selected from amongstthe substitutions and combinations of substitutions listed in each rowof FIG. 1.XXI. The phytase of any of embodiments I-XX, which has an improvedthermostability, an improved pH profile, an improved specific activity,an amended glycosylation pattern, an improved temperature profile, animproved performance in animal feed, and/or which incorporates a changeof a potential protease cleavage site.XXII. An isolated nucleic acid sequence comprising a nucleic acidsequence which encodes the phytase of any of embodiments I-XXI.XXIII. A nucleic acid construct comprising the nucleic acid sequence ofembodiment XXII operably linked to one or more control sequences thatdirect the production of the phytase in a suitable expression host.XXIV. A recombinant expression vector comprising the nucleic acidconstruct of embodiment XXIII.XXV. A recombinant host cell comprising the nucleic acid construct ofembodiment XXIII and/or the expression vector of embodiment XXIV.XXVI. A method for producing the phytase of any one of embodimentsI-XXI, comprising(a) cultivating the host cell of embodiment XXV to produce a supernatantcomprising the phytase; and (b) recovering the phytase.XXVII. A transgenic plant, or plant part, capable of expressing aphytase of any one of embodiments I-XXI.XXVIII. A transgenic, non-human animal, or products, or elementsthereof, being capable of expressing a phytase of any one of embodimentsI-XXI.XXIX. A composition comprising at least one phytase of any one ofembodiments I-XXI, and(a) at least one fat soluble vitamin;(b) at least one water soluble vitamin; and/or(c) at least one trace mineral.XXX. The composition of embodiment XXIX further comprising at least oneenzyme selected from the following group of enzymes: amylase, phytase,phosphatase, xylanase, galactanase, alpha-galactosidase, protease,phospholipase, and/or beta-glucanase.XXXI. The composition of any of embodiments XXIX-XXX which is an animalfeed additive.XXXII. An animal feed composition having a crude protein content of 50to 800 g/kg and comprising the phytase of any of embodiments I-XXI orthe composition of any of embodiments XXIX-XXXI.XXXIII. A method for improving the nutritional value of an animal feed,wherein the phytase of any one of embodiments I-XXI or the compositionof any one of embodiments XXIX-XXXI is added to the feed.XXXIV. A process for reducing phytate levels in animal manure comprisingfeeding an animal with an effective amount of the feed of embodimentXXXII.XXXV. A method for the treatment of vegetable proteins, comprising thestep of adding the phytase of any one of embodiments I-XXI or thecomposition of any one of embodiments XXIX-XXXI to at least onevegetable protein or protein source.XXXVI. Use of the phytase of any of embodiments I-XXI or the compositionof any of embodiments XXIX-XXXI in animal feed; in the preparation ofanimal feed; for improving the nutritional value of animal feed; forreducing phytate levels in animal manure; for the treatment of vegetableproteins; or for liberating phosphorous from a phytase substrate.a). A phytase which has at least 70% identity to SEQ ID NO: 2 and whichcomprises at least one alteration as compared to SEQ ID NO: 2 in atleast one position selected from the following: 1, 2, 3, 4, 5, 31, 41,46, 52, 53, 55, 57, 59, 74, 76, 82, 84, 91, 99, 100, 104, 105, 107, 109,111, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 136, 137,141, 154, 161, 162, 164, 167, 171, 176, 177, 179, 180, 181, 182, 183,184, 185, 186, 196, 199, 200, 202, 203, 218, 223, 239, 240, 241, 247,273, 276, 281, 282, 283, 284, 285, 286, 289, 294, 299, 308, 314, 316,324, 331, 339, 351, 355, 362, 379, 385, 406, 409, 410, and 411; with theproviso that the phytase is not SEQ ID NO: 3, not SEQ ID NO: 4, and notSEQ ID NO: 6.a1). A phytase which has at least 70% identity to SEQ ID NO: 2 and whichcomprises at least one alteration as compared to SEQ ID NO: 2 in atleast one position selected from the following: 1, 2, 3, 4, 5, 31, 41,46, 52, 53, 55, 57, 59, 74, 76, 82, 84, 91, 99, 100, 104, 105, 107, 109,111, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 136, 137,141, 154, 161, 162, 164, 167, 171, 176, 177, 179, 180, 181, 182, 183,184, 185, 186, 196, 199, 200, 202, 203, 218, 223, 239, 240, 241, 247,273, 276, 281, 282, 283, 284, 285, 286, 289, 294, 299, 308, 314, 316,324, 331, 339, 351, 355, 362, 379, 385, 406, 409, 410, and 411, with theproviso that the variant does not comprise (i) 31D/121T/316N/331E, andnot (ii) 31D/121N/316K/331E, and not (iii) 31N/121N/316N/331K.a2). A phytase which has at least 70% identity to SEQ ID NO: 2 and whichcomprises at least one alteration as compared to SEQ ID NO: 2 in atleast one position selected from the following: 1, 2, 3, 4, 5, 41, 46,52, 53, 55, 57, 59, 74, 76, 82, 84, 91, 99, 100, 104, 105, 107, 109,111, 114, 115, 116, 117, 118, 119, 120, 122, 123, 124, 136, 137, 141,154, 161, 162, 164, 167, 171, 176, 177, 179, 180, 181, 182, 183, 184,185, 186, 196, 199, 200, 202, 203, 218, 223, 239, 240, 241, 247, 273,276, 281, 282, 283, 284, 285, 286, 289, 294, 299, 308, 314, 324, 339,351, 355, 362, 379, 385, 406, 409, 410, and 411.a3). The phytase of embodiment a2), which comprises at least one of thefollowing alterations: 1*, 2*, 3*, 4P, 5P, 31C,T, 41P, 46C,D,E, 52C,E,53V,Q, 55I,D, 57Y, 59C, 74A, 76G, 82E, 84Y, 91C,P, 99C, 100C, 104A,105F, 107D,E,G, 109A,G, 111P, 114H,N,T, 115Q, 116A,E,P,T,Q 117D,E,K,118I,M,L,T, 119G,K,R,S, 120K,S,T,Q, 121A,D,M,P,V, 122D, 123P,S,124L,T,V, 136P, 137P, 141C, 154P, 161P, 162C, 164D,E, 167Q, 171T, 176C,177C, 179G,I,K,N,Q, 180A,E,G,T, 181D,G,I,K, 182H,K,S,Q, 183A,L,P,S,V,Q,184*, 185*, 186*, 196Q, 199C, 200K,R, 202N, 203T, 218Q, 223E, 239Q,240P, 241Q, 247C, 273L,Q, 276K,R, 281H, 282P, 283P, 284P, 285G,N,R,286K,Q, 289P, 294T, 299L, 308A, 314G,N, 316D, 324N, 339D, 351Y, 355P,362K,R, 379K,R, 385D, 406A, 409D,E, 410D,E, and/or 411K,R; and/orwherein the amino acids in position 179, 180, 181, 182, 183, 184, 185,and 186 have been replaced by QADKP (SEQ ID NO: 17), GEDKP (SEQ ID NO:18), NGISA (SEQ ID NO: 19), IAGKS (SEQ ID NO: 20), KEKHQ (SEQ ID NO:21), KEKQQ (SEQ ID NO: 22), KEKKV (SEQ ID NO: 23), or KTDKL (SEQ ID NO:24).a4). The phytase of any of embodiments a2)-a3), which comprises at leastone of the following alterations:(i) 31C, 46C, 52C, 59C, 91C, 99C, 100C, 141C, 162C, 176C, 177C, 199C,and/or 247C;(ii) 4P, 5P, 41P, 91P, 111P, 136P, 137P, 154P, 161P, 240P, 282P, 283P,284P, 289P, and/or 355P;(iii) 52E, 55D,I, 57Y, 76G, 84Y, 104A, 105F, 107D,G, 109A,G, 273L,Q,285G,R, 286Q, 294T, 299L, 351Y, and/or 362K;(iv) 1*, 1*/2*, or 1*/2*/3*;(v) wherein K179, T180, T181, E182, K183, S184, T185, and K186 have beenreplaced by QADKP (SEQ ID NO: 17), GEDKP (SEQ ID NO: 18), NGISA (SEQ IDNO: 19), IAGKS (SEQ ID NO: 20), KEKHQ (SEQ ID NO: 21), KEKQQ (SEQ ID NO:22), KEKKV (SEQ ID NO: 23), or KTDKL (SEQ ID NO: 24);(vi) 119K,R, and/or 411K,R;(vii) 107E, and/or 164D,E;(viii) 46D,E, 82E, 223E, 276K,R, 362K,R, 379K,R, 385D, 409D,E, and/or410D,E;(ix) 53V,Q, 121D, 167Q, 196Q, 200K,R, 202N, 218Q, 239Q, 241Q, 285N,314G,N, 324N, and/or 406A;(x) 114H,N,T 115Q, 116A,E,P,T,Q, 117D,E,K, 118I,L,M,T 119G,K,S,120K,S,T,Q, 121A,M,P,V, 122D, 123P,S, and/or 124L,T,V(xi) 31T, 74A, 171T, 203T, 281H, 308A, and/or 316D; and/or(xii) 339D.a5). The phytase of any of embodiments a2)-a4), which comprises at leastone of the following alterations:(i) 141C/199C, 910/460, 52C/99C, 31C/176C, 31C/177C, 59C/100C, and/or162C/247C;(ii) 41P, 91P, 136P, 137P, 154P, 161P, 355P, 111P, 240P, 282P, 283P,284P, 289P, 4P, and/or 5P;(iii) 52E, 55I, 57Y, 104A/105F, 107D,G, 109A,G, 76G, 84Y, 362K, 273L,Q,285G,R, 286Q, 294T, 299L, 331K/55D, and/or 351Y;(iv) 1*, 1*/2*, or 1*/2*/3*;(v) wherein K179, T180, T181, E182, K183, S184, T185, and K186 have beenreplaced by QADKP (SEQ ID NO: 17), GEDKP (SEQ ID NO: 18), NGISA (SEQ IDNO: 19), IAGKS (SEQ ID NO: 20), KEKHQ (SEQ ID NO: 21), KEKQQ (SEQ ID NO:22), KEKKV (SEQ ID NO: 23), or KTDKL (SEQ ID NO: 24);(vi) 119R, K, and/or 411R,K;(vii) 107E, and/or 164E,D;(viii) 362R,K, 276R,K, 379R,K, 409D,E, 223E, 385D, 46D,E, 410D,E, and/or82E;(ix) 218Q, 324N, 200R,K, 121D, 196Q, 202N, 406A, 167Q, 53V,Q, 241Q,314N,G, 239Q, and/or 285N;(x) 114H/115Q/116E/117K/118M/119G/120T/121M/122D/123P/124T,114H/115Q/116Q/117D/118I/119K/120Q/121V/122D/123S/124L,114H/115Q/116P/117E/118I/119G/120K/121M/122D/123P/124V,114T/115Q/116A/117D/118T/119S/120S/121P/122D/123P/124L,114H/115Q/116Q/117D/118I/119K/120Q/121A/122D/123P/124L,114T/115Q/116T/117D/118T/119S/120S/121P/122D/123P/124L, or114N/115Q/116A/117D/118L/119K/120K/121T/122D/123P/124L;(xi) 31T, 74A, 171T, 203T, 281H, 308A, and/or 316D; and/or(xii) 339D.b). The phytase of embodiment a) or a1), which comprises at least one ofthe following alterations: 1*, 2*, 3*, 4P, 5P, 31C,T, 41P, 46C,D,E,52C,E, 53V,Q, 55D,I, 57Y, 59C, 74A, 76G, 82E, 84Y, 91C,P, 99C, 100C,104A, 105F, 107D,E,G, 109A,G, 111P, 114H,N,T, 115Q, 116A,E,P,T,Q,117D,E,K 118I,L,M,T 119G,K,R,S, 120K,S,T,Q, 121A,D,M,P,T,V, 122D,123P,S, 124L,T,V, 136P, 137P, 141C, 154P, 161P, 162C, 164D,E, 167Q,171T, 176C, 177C, 179G,I,K,N,Q, 180A,E,G,T, 181D,G,I,K, 182H,K,S,Q,183A,L,P,S,V,Q, 184*, 185*, 186*, 196Q, 199C, 200K,R, 202N, 203T, 218Q,223E, 239Q, 240P, 241Q, 247C, 273L,Q, 276K,R, 281H, 282P, 283P, 284P,285G,N,R, 286K,Q, 289P, 294T, 299L, 308A, 314G,N, 316D, 324N, 331K,339D, 351Y, 355P, 362K,R, 379K,R, 385D, 406A, 409D,E, 410D,E, and/or411R,K; and/or wherein the amino acids in position 179, 180, 181, 182,183, 184, 185, and 186 have been replaced by QADKP (SEQ ID NO: 17),GEDKP (SEQ ID NO: 18), NGISA (SEQ ID NO: 19), IAGKS (SEQ ID NO: 20),KEKHQ (SEQ ID NO: 21), KEKQQ (SEQ ID NO: 22), KEKKV (SEQ ID NO: 23), orKTDKL (SEQ ID NO: 24).c). The phytase of any of the above embodiments, which comprises atleast one of the following alterations:(i) 31C, 46C, 52C, 59C, 91C, 99C, 100C, 141C, 162C, 176C, 177C, 199C,and/or 247C;(ii) 4P, 5P, 41P, 91P, 111P, 136P, 137P, 154P, 161P, 240P, 282P, 283P,284P, 289P, and/or 355P;(iii) 52E, 55D,I, 57Y, 76G, 84Y, 104A, 105F, 107D,G, 109A,G, 121T,273L,Q, 285G,R, 286Q, 294T, 299L, 331K, 351Y, and/or 362K;(iv) 1*, 1*/2*, or 1*/2*/3*;(v) wherein K179, T180, T181, E182, K183, S184, T185, and K186 have beenreplaced by QADKP (SEQ ID NO: 17), GEDKP (SEQ ID NO: 18), NGISA (SEQ IDNO: 19), IAGKS (SEQ ID NO: 20), KEKHQ (SEQ ID NO: 21), KEKQQ (SEQ ID NO:22), KEKKV (SEQ ID NO: 23), or KTDKL (SEQ ID NO: 24);(vi) 119K,R, and/or 411K,R;(vii) 107E, and/or 164D,E;(viii) 46D,E, 82E, 223E, 276K,R, 362K,R, 379K,R, 385D, 409D,E, and/or410D,E;(ix) 53V,Q, 121D, 167Q, 196Q, 200K,R, 202N, 218Q, 239Q, 241Q, 285N,314G,N, 324N, and/or 406A;(x) 114H,N,T 115Q, 116A,E,P,T,Q, 117D,E,K, 118I,L,M,T 119G,K,S,120K,S,T,Q, 121A,M,P,T,V, 122D, 123P,S, and/or 124L,T,V;(xi) 31T, 74A, 171T, 203T, 281H, 308A, and/or 316D; and/or(xii) 339D.c1). The phytase of any of the above embodiments, which comprises atleast one of the following alterations:(i) 31C, 46C, 52C, 59C, 91C, 99C, 100C, 141C, 162C, 176C, 177C, 199C,and/or 247C, preferably 52C, 99C, 141C, and/or 199C;(ii) 4P, 5P, 41P, 91P, 111P, 136P, 137P, 154P, 161P, 240P, 282P, 283P,284P, 289P, and/or 355P, preferably 4P, 5P, 111P;(iii) 52E, 55D,I, 57Y, 76G, 84Y, 104A, 105F, 107D,G, 109A,G, 121T,273L,Q, 285G,R, 286Q, 294T, 299L, 331K, 351Y, and/or 362K, preferably57Y, 76G, 107G, 273L, 286Q and/or 362K;(iv) 1*, 1*/2*, or 1*/2*/3*;(v) wherein K179, T180, T181, E182, K183, S184, T185, and K186 have beenreplaced by QADKP (SEQ ID NO: 17), GEDKP (SEQ ID NO: 18), NGISA (SEQ IDNO: 19), IAGKS (SEQ ID NO: 20), KEKHQ (SEQ ID NO: 21), KEKQQ (SEQ ID NO:22), KEKKV (SEQ ID NO: 23), or KTDKL (SEQ ID NO: 24), preferably KEKKV(SEQ ID NO: 23);(vi) 119K,R, and/or 411K,R, preferably 119K;(vii) 107E, and/or 164D,E;(viii) 46D,E, 82E, 223E, 276K,R, 362K,R, 379K,R, 385D, 409D,E, and/or410D,E preferably 46E, 223E 362K,R, and/or 379K,R;(ix) 53V,Q, 121D, 167Q, 196Q, 200K,R, 202N, 218Q, 239Q, 241Q, 285N,314G,N, 324N, and/or 406A, preferably 53V, 121D, 196Q, 200K, 202N, 218Q,241Q, 314N, and/or 406A;(x) 114H,N,T 115Q, 116A,E,P,T,Q, 117D,E,K, 118I,L,M,T 119G,K,S,120K,S,T,Q, 121A,M,P,T,V, 122D, 123P,S, and/or 124L,T,V preferably 114T115Q, 116A,T, 117D, 118T 119K,S, 120S, 121P, 122D, 123P, and/or 124L;(xi) 31T, 74A, 171T, 203T, 281H, 308A, and/or 316D; and/or(xii) 339D.d). The phytase of any of the above embodiments, which has improvedproperties.e). The phytase of embodiment c) or c1), which comprises at least one ofthe one or more alterations of features (i), (ii), (iii), (iv), (v),(vi), (vii), (viii), (x), (xi) and/or (xii) of embodiment 3 and has animproved thermostability.f). The phytase of embodiment c) or c1), which comprises at least one ofthe one or more alterations of features (ix) and/or (x) of embodiment c)and has an improved pH profile.g). The phytase of embodiment c) or c1), which comprises at least one ofthe one or more alterations of feature (x) of embodiment c) and has animproved specific activity.h). The phytase of embodiment c) or c1), which comprises at least one ofthe one or more alterations of feature (xi) of embodiment c) and has anamended glycosylation pattern.i). The phytase of embodiment c) or c1), which comprises the alterationof feature (xii) of embodiment c) which changes a potential proteasecleavage site.j). The phytase of any one of embodiment a)-d) including a1)-a5) andc1), which comprises at least one of the following alterations:(i) 141C/199C, 910/460, 520/990, 31C/176C, 31C/177C, 590/1000, and/or1620/2470;(ii) 41P, 91P, 136P, 137P, 154P, 161P, 355P, 111P, 240P, 282P, 283P,284P, 289P, 4P, and/or 5P;(iii) 52E, 55I, 57Y, 104A/105F, 107D,G, 109A,G, 76G, 84Y, 121T, 362K,273L,Q, 285G,R, 286Q, 294T, 299L, 331K/55D, and/or 351Y;(iv) 1*, 1*/2*, or 1*/2*/3*;(v) wherein K179, T180, T181, E182, K183, S184, T185, and K186 have beenreplaced by QADKP (SEQ ID NO: 17), GEDKP (SEQ ID NO: 18), NGISA (SEQ IDNO: 19), IAGKS (SEQ ID NO: 20), KEKHQ (SEQ ID NO: 21), KEKQQ (SEQ ID NO:22), KEKKV (SEQ ID NO: 23), or KTDKL (SEQ ID NO: 24);(vi) 119R, K, and/or 411R,K;(vii) 107E, and/or 164E,D;(viii) 362R,K, 276R,K, 379R,K, 409D,E, 223E, 385D, 46D,E, 410D,E, and/or82E;(ix) 218Q, 324N, 200R,K, 121D, 196Q, 202N, 406A, 167Q, 53V,Q, 241Q,314N,G, 239Q, and/or 285N;(x) 114H/115Q/116E/117K/118M/119G/120T/121M/122D/123P/124T,114H/115Q/116Q/117D/118I/119K/120Q/121V/122D/123S/124L,114H/115Q/116P/117E/118I/119G/120K/121M/122D/123P/124V,114T/115Q/116A/117D/118T/119S/120S/121P/122D/123P/124L,114H/115Q/116Q/117D/118I/119K/120Q/121A/122D/123P/124L,114T/115Q/116T/117D/118T/119S/120S/121P/122D/123P/124L, or114N/115Q/116A/117D/118L/119K/120K/121T/122D/123P/124L;(xi) 31T, 74A, 171T, 203T, 281H, 316D, and/or 308A; and/or(xii) 339D.k). The phytase of any one of embodiment a)-d) including a1)-a5) andc1), which comprises at least one of the following alterations:(i) K141C/V199C, Q91C/W46C, G52C/A99C, N31C/E176C, N31C/T177C,G59C/F100C, and/or S162C/S247C;(ii) D41P, Q91P, N136P, T137P, L154P, S161P, T355P, Q111P, K240P, G282P,T283P, T284P, G289P, N4P, and/or GSP;(iii) G52E, V55I, E57Y, L104A/A105F, K107D,G, Q109A,G, T76G, A84Y,N121T, 1362K, M273L,Q, E285G,R, N286Q, V294T, 1299L, E331K/V55D, and/orF351Y;(iv) E1*, E1*/E2*, or E1*/E2*/Q3*;(v) wherein K179, T180, T181, E182, K183, S184, T185, and K186 have beenreplaced by QADKP (SEQ ID NO: 17), GEDKP (SEQ ID NO: 18), NGISA (SEQ IDNO: 19), IAGKS (SEQ ID NO: 20), KEKHQ (SEQ ID NO: 21), KEKQQ (SEQ ID NO:22), KEKKV (SEQ ID NO: 23), or KTDKL (SEQ ID NO: 24);(vi) E119R,K, and/or E411R,K;(vii) K107E, and/or R164E,D;(viii) 1362R,K, T276R,K, I379R,K, V409D,E, Q223E, N385D, W46D,E,T410D,E, and/or Q82E;(ix) E218Q, D324N, T200R,K, N121D, E196Q, D202N, E406A, E167Q, E53V,Q,E241Q, D314N,G, E239Q, and/or E285N;(x) Y114H/K116E/D117K/E118M/E119G/K120T/N121M/L124T,Y114H/K116Q/E118I/E119K/K120Q/N121V/P123S,Y114H/K116P/D117E/E118I/E119G/N121M/L124V,Y114T/K116A/E118T/E119S/K120S/N121P,Y114H/K116Q/E118I/E119K/K120Q/N121A,Y114T/K116T/E118T/E119S/K120S/N121P, or Y114N/K116A/E118L/E119K/N121T;(xi) N31T, N74A, N171T, N203T, N281H, N316D, and/or N308A; and/or(xii) R339D.l). The phytase of embodiment k) which is a variant of SEQ ID NO: 2.m). The phytase of any one of embodiment a)-d) including a1)-a5) andc1), which comprises at least one of the following alterations:(i) T141C/V199C, Q91C/W46C, G52C/A99C, D31C/E176C, D31C/T177C,G59C/F100C, and/or S162C/S247C;(ii) D41P, Q91P, N136P, T137P, L154P, S161P, T355P, Q111P, K240P, G282P,T283P, T284P, G289P, N4P, and/or G5P;(iii) G52E, V55I, E57Y, L104A/A105F, K107D,G, Q109A,G, T76G, A84Y,1362K, M273L,Q, E285G,R, N286Q, V294T, 1299L, E331K/V55D, and/or F351Y;(iv) E1*, E1*/E2*, or E1*/E2*/Q3*;(v) wherein K179, T180, T181, E182, K183, S184, T185, and K186 have beenreplaced by QADKP (SEQ ID NO: 17), GEDKP (SEQ ID NO: 18), NGISA (SEQ IDNO: 19), IAGKS (SEQ ID NO: 20), KEKHQ (SEQ ID NO: 21), KEKQQ (SEQ ID NO:22), KEKKV (SEQ ID NO: 23), or KTDKL (SEQ ID NO: 24);(vi) E119R,K, and/or E411R,K;(vii) K107E, R164E,D;(viii) 1362R,K, T276R,K, I379R,K, V409D,E, Q223E, N385D, W46D,E,T410D,E, Q82E;(ix) E218Q, D324N, T200R,K, T121D, E196Q, D202N, E406A, E167Q, E53V,Q,E241Q, D314N,G, E239Q, and/or E285N;(x) Y114H/K116E/D117K/E118M/E119G/K120T/T121M/L124T,Y114H/K116Q/E118I/E119K/K120Q/T121V/P123S,Y114H/K116P/D117E/E118I/E119G/T121M/L124V,Y114T/K116A/E118T/E119S/K120S/T121P/,Y114H/K116Q/E118I/E119K/K120Q/T121A/,Y114T/K116T/E118T/E119S/K120S/T121P, or Y114N/K116A/E118L/E119K;(xi) N74A, N171T, N203T, N281H, N316D, and/or N308A; and/or(xii) R339D.n). The phytase of embodiment m) which is a variant of SEQ ID NO: 4.o). The phytase of any one of embodiment a)-d) including a1)-a5) andc1), which comprises at least one of the following alterations:(i) K141C/V199C, Q91C/W46C, G52C/A99C, D31C/E176C, D31C/T177C,G59C/F100C, and/or S162C/S247C;(ii) D41P, Q91P, N136P, T137P, L154P, S161P, T355P, Q111P, K240P, G282P,T283P, T284P, G289P, N4P, and/or G5P;(iii) G52E, V55I, E57Y, L104A/A105F, K107D,G, Q109A,G, T76G, A84Y,N121T, 1362K, M273L,Q, E285G,R, N286Q, V294T, 1299L, E331K/V55D, and/orF351Y;(iv) E1*, E1*/E2*, or E1*/E2*/Q3*;(v) wherein K179, T180, T181, E182, K183, S184, T185, and K186 have beenreplaced by QADKP (SEQ ID NO: 17), GEDKP (SEQ ID NO: 18), NGISA (SEQ IDNO: 19), IAGKS (SEQ ID NO: 20), KEKHQ (SEQ ID NO: 21), KEKQQ (SEQ ID NO:22), KEKKV (SEQ ID NO: 23), or KTDKL (SEQ ID NO: 24);(vi) E119R,K, and/or E411R,K;(vii) K107E, and/or R164E,D;(viii) 1362R,K, T276R,K, I379R,K, V409D,E, Q223E, N385D, W46D,E,T410D,E, and/or Q82E;(ix) E218Q, D324N, T200R,K, N121D, E196Q, D202N, E406A, E167Q, E53V,Q,E241Q, D314N,G, E239Q, and/or E285N;(x) Y114H/K116E/D117K/E118M/E119G/K120T/N121M/L124T,Y114H/K116Q/E118I/E119K/K120Q/N121V/P123S,Y114H/K116P/D117E/E118I/E119G/N121M/L124V,Y114T/K116A/E118T/E119S/K120S/N121P,Y114H/K116Q/E118I/E119K/K120Q/N121A,Y114T/K116T/E118T/E119S/K120S/N121P, or Y114N/K116A/E118L/E119K/N121T;(xi) N74A, N171T, N203T, N281H, and/or N308A; and/or(xii) R339D.p). The phytase of embodiment o) which is a variant of SEQ ID NO: 3.q). The phytase of any one of embodiment a)-d) including a1)-a5) andc1), which comprises at least one of the following alterations:(i) K141C/V199C, Q91C/W46C, G52C/A99C, N31C/E176C, N31C/T177C,G59C/F100C, and/or S162C/S247C;(ii) D41P, Q91P, N136P, T137P, L154P, S161P, T355P, Q111P, K240P, G282P,T283P, T284P, G289P, N4P, and/or G5P;(iii) G52E, V55I, E57Y, L104A/A105F, K107D,G, Q109A,G, T76G, A84Y,N121T, 1362K, M273L,Q, E285G,R, N286Q, V294T, 1299L, V55D, and/or F351Y;(iv) E1*, E1*/E2*, or E1*/E2*/Q3*;(v) wherein K179, T180, T181, E182, K183, S184, T185, and K186 have beenreplaced by QADKP (SEQ ID NO: 17), GEDKP (SEQ ID NO: 18), NGISA (SEQ IDNO: 19), IAGKS (SEQ ID NO: 20), KEKHQ (SEQ ID NO: 21), KEKQQ (SEQ ID NO:22), KEKKV (SEQ ID NO: 23), or KTDKL (SEQ ID NO: 24);(vi) E119R,K, and/or E411R,K;(vii) K107E, and/or R164E,D;(viii) 1362R,K, T276R,K, I379R,K, V409D,E, Q223E, N385D, W46D,E,T410D,E, and/or Q82E;(ix) E218Q, D324N, T200R,K, N121D, E196Q, D202N, E406A, E167Q, E53V,Q,E241Q, D314N,G, E239Q, and/or E285N;(x) Y114H/K116E/D117K/E118M/E119G/K120T/N121M/L124T,Y114H/K116Q/E118I/E119K/K120Q/N121V/P123S,Y114H/K116P/D117E/E118I/E119G/N121M/L124V,Y114T/K116A/E118T/E119S/K120S/N121P,Y114H/K116Q/E118I/E119K/K120Q/N121A,Y114T/K116T/E118T/E119S/K120S/N121P, or Y114N/K116A/E118L/E119K/N121T;(xi) N31T, N74A, N171T, N203T, N281H, N316D, and/or N308A; and/or(xii) R339D.r). The phytase of embodiment q) which is a variant of SEQ ID NO: 6.s). The phytase of any one of embodiment a)-d) including a1)-a5) andc1), which comprises at least one of the following alterations:(i) K141C/V199C, Q91C/W46C, G52C/A99C, D31C/E176C, D31C/T177C,G59C/F100C, and/or S162C/S247C;(ii) D41P, Q91P, N136P, T137P, L154P, S161P, T355P, Q111P, K240P, G282P,T283P, T284P, G289P, N4P, and/or G5P;(iii) G52E, V55I, E57Y, L104A/A105F, K107D,G, Q109A,G, T76G, A84Y,1362K, M273L,Q, E285G,R, N286Q, V294T, 1299L, E331K/V55D, and/or F351Y;(iv) E1*, E1*/E2*, or E1*/E2*/P3*;(v) wherein K179, T180, T181, E182, K183, S184, T185, and K186 have beenreplaced by QADKP (SEQ ID NO: 17), GEDKP (SEQ ID NO: 18), NGISA (SEQ IDNO: 19), IAGKS (SEQ ID NO: 20), KEKHQ (SEQ ID NO: 21), KEKQQ (SEQ ID NO:22), KEKKV (SEQ ID NO: 23), or KTDKL (SEQ ID NO: 24);(vi) E119R,K, and/or E411R,K;(vii) K107E, and/or R164E,D;(viii) 1362R, K, T276R,K, I379R, K, V409D,E, Q223E, N385D, W46D,E,T410D,E, and/or Q82E;(ix) E218Q, D324N, T200R,K, T121D, E196Q, D202N, E406A, E167Q, E53V,Q,E241Q, D314N,G, E239Q, and/or E285N;(x) Y114H/K116E/D117K/E118M/E119G/K120T/T121M/L124T,Y114H/K116Q/E118I/E119K/K120Q/T121V/P123S,Y114H/K116P/D117E/E118I/E119G/T121M/L124V,Y114T/K116A/E118T/E119S/K120S/T121P,Y114H/K116Q/E118I/E119K/K120Q/T121A,Y114T/K116T/E118T/E119S/K120S/T121P, or Y114N/K116A/E118L/E119K;(xi) D31T, N74A, N171T, N203T, N281H, N316D, and/or N308A; and/or(xii) R339D.t). The phytase of embodiment s) which is a variant of SEQ ID NO: 9.u). An isolated nucleic acid sequence comprising a nucleic acid sequencewhich encodes the phytase of any of embodiment a)-t) including a1)-a5)and c1).v). A nucleic acid construct comprising the nucleic acid sequence ofembodiment u) operably linked to one or more control sequences thatdirect the production of the phytase in a suitable expression host.w). A recombinant expression vector comprising the nucleic acidconstruct of embodiment v).x). A recombinant host cell comprising the nucleic acid construct ofembodiment v) and/or the expression vector of embodiment w).y). A method for producing the phytase of any one of embodiment a)-t)including a1)-a5) and c1), comprising(a) cultivating the host cell of embodiment x) to produce a supernatantcomprising the phytase; and (b) recovering the phytase.z). A transgenic plant, or plant part, capable of expressing a phytaseof any one of embodiment a)-t) including a1)-a5) and c1).ae). A transgenic, non-human animal, or products, or elements thereof,being capable of expressing a phytase of any one of embodiment a)-t)including a1)-a5) and c1).oe). A composition comprising at least one phytase of any one ofembodiment a)-t) includinga1)-a5) and c1), and(a) at least one fat soluble vitamin;(b) at least one water soluble vitamin; and/or(c) at least one trace mineral.aa). The composition of embodiment oe) further comprising at least oneenzyme selected from the following group of enzymes: amylase, phytase,phosphatase, xylanase, galactanase, alpha-galactosidase, protease,phospholipase, and/or beta-glucanase.bb). The composition of any one of embodiment oe)-aa) which is an animalfeed additive.cc). An animal feed composition having a crude protein content of 50 to800 g/kg and comprising the phytase of any one of embodiment a)-t)including a1)-a5) and c1) or the composition of any one of embodimentoe)-aa).dd). A method for improving the nutritional value of an animal feed,wherein the phytase of any one of embodiment a)-t) including a1)-a5) andc1) or the composition of any one of embodiment oe)-aa) is added to thefeed.ee). A process for reducing phytate levels in animal manure comprisingfeeding an animal with an effective amount of the feed of embodimentcc).ff). A method for the treatment of vegetable proteins, comprising thestep of adding the phytase of any one of embodiment a)-t) includinga1)-a5) and c1) or the composition of any one of embodiment oe)-aa) toat least one vegetable protein or protein source.gg). Use of the phytase of any one of embodiment a)-t) including a1)-a5)and c1) or the composition of any one of embodiment oe)-aa) in animalfeed; in the preparation of animal feed; for improving the nutritionalvalue of animal feed; for reducing phytate levels in animal manure; forthe treatment of vegetable proteins; or for liberating phosphorous froma phytase substrate.

The invention described and claimed herein is not to be limited in scopeby the specific embodiments herein disclosed, since these embodimentsare intended as illustrations of several aspects of the invention. Anyequivalent embodiments are intended to be within the scope of thisinvention. Indeed, various modifications of the invention in addition tothose shown and described herein will become apparent to those skilledin the art from the foregoing description. Such modifications are alsointended to fall within the scope of the appended claims. In the case ofconflict, the present disclosure including definitions will control.

Various references are cited herein, the disclosures of which areincorporated by reference in their entireties.

EXAMPLES

Chemicals used were commercial products of at least reagent grade.

Example 1: Preparation of Variants, and Test of Thermostability and pHProfile

Preparation of Phytase Variants

DNA encoding a variant of the phytase having the amino acid sequence ofSEQ ID NO: 2 is generated by methods known in the art, and theconstructs are fused by PCR to the DNA coding for the signal peptidedescribed by Takami et al., 1992, Biosci. Biotechnol. Biochem. 56:1455and integrated by homologous recombination into the genome of a Bacillussubtilis host cell (see Diderichsen et al., 1990, J. Bacteriol. 172:4315-4321) using standard techniques. The genes are expressed under thecontrol of a triple promoter system (as described in WO 99/43835) andthe resulting phytase proteins purified using conventional methods.

Determination of Temperature Stability

The temperature stability of a phytase variant may be determined in thefollowing way: 500 microliter protein solution of the variant and of thereference protein (SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, and/or SEQID NO: 6) having approximately 10 microgram protein per ml, and beingdissolved in 0.1 M Na-acetate buffer, pH 5.5, are split in two portions,one portion is incubated at a desired elevated temperature (e.g., 50°C., 55° C., 60° C., 65° C., 70° C., 75° C., 80° C., or 85° C.) inplastic containers, the other is stored at 5° C. After 30 minutesincubation at the elevated temperature the protein solutions aretransferred to an ice-bath and the activity of the cooled as well as theheated sample is measured by the phosphatase assay described below(buffer blind subtracted). The residual activity is defined as theactivity after heat-treatment divided by the activity of the cooledsample (in %). A variant is considered to be more temperature stable(thermostable) if the residual activity in the phosphatase, or phytase,assay is higher, as compared to the reference.

Determination of Phosphatase Activity

75 microliters phytase-containing enzyme solution is dispensed in amicrotiter plate well, e. g. NUNC 269620 and 75 microliter substrate isadded (for preparing the substrate, two 5 mg p-nitrophenyl phosphatetablets (Sigma, Cat. No. N-9389) are dissolved in 10 ml 0.1 M Na-acetatebuffer, pH 5.5). The plate is sealed and incubated 15 min., shaken with750 rpm at 37° C. After the incubation time 75 microliter stop reagentis added (the stop reagent is 0.1 M di-sodium tetraborate in water) andthe absorbance at 405 nm is measured in a microtiter platespectrophotometer. One phosphatase unit is defined as the enzymeactivity that releases 1 micromol phosphate/min under the given reactionconditions (buffer blind subtracted). The absorbance of 1 micromolp-nitrophenol is determined to be 56 AU (AU=absorbancy units) underassay conditions.

DSC Measurements

Differential Scanning calorimetry (DSC) may be performed at variouspH-values using the VP-DSC from Micro Cal. Scans are performed at aconstant scan rate of 1.5° C./min from 20-90° C. Before running the DSC,the phytases are desalted using NAP-5 columns (Pharmacia) equilibratedin the appropriate buffers (e.g., 0.1 M glycine-HCl, pH 2.5 or 3.0; 20mM sodium acetate pH 4.0; 0.1 M sodium acetate, pH 5.5; 0.1 M Tris-HCl,pH 7.0). Data-handling is performed using the MicroCal Origin software(version 4.10), and the denaturation temperature, Td (also called themelting temperature, Tm) is defined as the temperature at the apex ofthe peak in the thermogram.

Amended pH Profile: Determination of pH 3.5/5.5 Activity Ratio

An amendment of the pH profile of a phytase variant may be determined asfollows: The activity is measured at pH 3.5 (0.1 M acetate buffer, pH3.5) and at pH 5.5 (0.1 M acetate buffer, pH 5.5), in both cases thebuffer blind is subtracted. The activity determined at pH 3.5 is dividedby the activity determined at pH 5.5, i.e., the two absorbancymeasurements are divided (see below). To measure the activity,supernatants of the variants and references are appropriately diluted(e.g., 1:5000) in the respective buffer. 75 microliter of the respectiveenzyme solution is dispensed in a microtiter plate well, e. g. NUNC269620 and 75 microliter substrate with the corresponding pH is added(the substrate is prepared by dissolving two 5 mg p-nitrophenylphosphate tablets (Sigma, Cat. No. N-9389) in 10 ml 0.1 M Na-acetatebuffer, pH 5.5 and 10 ml 0.1 M acetate buffer, pH 3.5, respectively).The plate is sealed and incubated 15 min., shaken with 750 rpm at 37° C.After the incubation time 75 microliter stop (0.1 M di-sodiumtetraborate in water) reagent is added and the absorbance at 405 nm ismeasured in a microtiter plate spectrophotometer.

Determination of Phytase Activity

75 microliters phytase-containing enzyme solution, appropriately diluted(e.g., in 0.25 M sodium acetate, 0.005% (w/v) Tween-20. pH 5.5), isdispensed in a microtiter plate well, e.g., NUNC 269620, and 75microliter substrate is added (prepared by dissolving 100 mg sodiumphytate from rice (Aldrich Cat. No. 274321) in 10 ml 0.25 M Na-acetatebuffer, pH 5.5). The plate is sealed and incubated 15 min. shaken with750 rpm at 37° C. After the incubation time 75 microliter stop reagentis added (the stop reagent being prepared by mixing 10 ml molybdatesolution (10% (w/v) Ammonium hepta-molybdate in 0.25% (w/v) ammoniasolution); 10 ml ammonium vanadate (0.24% commercial product fromBie&Berntsen, Cat. No. LAB17650) and 21.7% (w/v) nitric acid) theabsorbance at 405 nm is measured in a microtiter platespectrophotometer. The phytase activity is expressed in the unit of FYT,one FYT being the amount of enzyme that liberates 1 micromol inorganicortho-phosphate per min. under the conditions above. An absolute valuefor the measured phytase activity is obtained by reference to a standardcurve prepared from appropriate dilutions of inorganic phosphate or to astandard curve made from dilutions of a phytase enzyme preparation withknown activity (such standard enzyme preparation with a known activityis available on request from Novozymes A/S, Krogshoejvej 36, DK-2880Bagsvaerd).

Example 2: Influence of Expression Host/Glycosylation on Thermostability

Expression in Bacillus

The phytase of SEQ ID NO: 2 was expressed in Bacillus subtilis asdescribed in Example 1, and purified using conventional methods:Centrifugation, germ filtration, ammonium sulphate precipitation (80%ammonium sulphate saturation), centrifugation, re-suspension of pelletsin buffer A (50 mM sodium acetate, 1.5 M ammonium sulphate pH 4.5),filtration, hydrophobic interaction chromatography (Phenyl Toyopearl,loading with buffer A, eluting with buffer B (50 mM sodium acetate pH4.5)), and cation exchange chromatography (SP-sepharose, loading with 10mM sodium citrate pH 4.0, eluting with a linear salt gradient (10 mMsodium citrate pH 4.0+1 M NaCl).

Expression in Pichia

Still further, the phytase of SEQ ID NO: 2 was expressed in Pichiapastoris as generally described by Rodriguez et al., 2000, Archives ofBiochemistry and Biophysics 382(1): 105-112. The phytase was purifiedfrom the supernatant of the fermentation broth as follows: Precipitationwith ammonium sulfate (80% saturation), re-dissolution in 10 ml 25 mMsodium acetate buffer Ph 4.5, dialysis against the same buffer, andfiltration through a 0.45 mm filter. 150 ml of this solution was appliedto a 40 ml SP Sepharose FF column (Pharmacia) equilibrated with the samebuffer pH 4.5, and the protein was eluted with a linear NaCl gradient(0-0.5 M). Fractions from the column were analyzed for phytase activity.Fractions with phytase activity were checked by SDS-PAGE and the purefractions were pooled. The protein concentration was measured by usingBCA kit (Pierce).

Thermostability by DSC

The Pichia- and the Bacillus-expressed phytase of SEQ ID NO: 2 weresubjected to thermostability measurements by Differential Scanningcalorimetry (DSC).

Sample Preparation:

Samples (less than 3 ml in volume) were dialyzed in a cold room (approx.5° C.) for a minimum of 1 hour against 500 ml of 20 mM sodium acetatebuffer pH 4.0. The sample was transferred to 500 ml of fresh, coldbuffer preparation and left to dialyze overnight. The samples werefiltered using a 0.45 micrometer syringe filter, volume adjusted toapprox. 1.5 ml using the dialysis buffer, and A₂₈₀ (absorbancy at 280nm) recorded. The dialysis buffer was used as reference in the DSCscans. The samples were degassed using vacuum suction and stirring forapprox. 10 minutes.

During sample preparation of the Pichia-expressed phytase (dialysisagainst 20 mM sodium acetate (NaAc) pH 4.0) a precipitate was formed.The supernatant was used for a first experiment. Afterwards theremaining part of the purified stock solution was dialysed against 20 mMNaAc pH 4.0 and this allowed precipitation of some low Mw impuritiespresent in the batch. This batch was used for a second experiment whichrevealed a Tm very similar to the first experiment (54 vs. 55° C.).

DSC Experiment:

Experimental settings using a MicroCal™ VP-DSC instrument: Scan rate: 90K/h. Scan interval: 20-90° C. Feedback mode: None. Filtering period: 16s.

The enzyme concentrations of the samples were approx. 1-1.5 mg/ml asestimated by A₂₈₀ and a theoretically calculated extinction coefficientat 280 nm (Vector NTI version 9.0.0). The thermal unfolding temperature(Td) was evaluated using MicroCal Origin software (version 4.10) and thedenaturation temperature determined as the temperature at the apex inthe thermogram.

The results are summarized in Table 2 below.

TABLE 2 Scanrate Scan interval T_(d) Host cell Buffer (° C./h) (° C.) (°C.) A₂₈₀ B. subtilis 20 mM NaAc pH 4.0 90 20-90 62 1.6 P. pastoris 20 mMNaAc pH 4.0 90 20-90 55 1.9

From Table 2 it clearly appears that the Pichia-expressed phytase ismuch less thermostable than the Bacillus-expressed phytase.

The Pichia-expressed phytase was heavily glycosylated, as visualized bya broad range of molecular masses using mass spectrometry (Maldi-TOF),whereas the Bacillus-expressed phytase was not glycosylated.

Example 3: Phytase Variant R339D

A protein-engineered variant of the phytase of SEQ ID NO: 2 having thesubstitution R339D was prepared and expressed in Aspergillus oryzaeusing methods known in the art. Its denaturation temperature, Td, wasdetermined to 62.5° C. (20 mM sodium acetate, pH 4.0), using DSC asdescribed in Example 2.

The R339D substitution furthermore serves to remove a Kex2 proteasecleavage site of potential relevance for expression in Aspergillus.

Example 4: Animal Feed and Animal Feed Additives Comprising a PhytaseVariant

Animal Feed Additive

A formulation of phytase variant R339D of SEQ ID NO: 2 containing 0.15 gphytase enzyme protein is added to the following premix (per kilo ofpremix):

5000000 IE Vitamin A 1000000 IE Vitamin D3 13333 mg Vitamin E 1000 mgVitamin K3 750 mg Vitamin B1 2500 mg Vitamin B2 1500 mg Vitamin B6 7666mcg Vitamin B12 12333 mg Niacin 33333 mcg Biotin 300 mg Folic Acid 3000mg Ca-D-Panthothenate 1666 mg Cu 16666 mg Fe 16666 mg Zn 23333 mg Mn 133mg Co 66 mg I 66 mg Se 5.8 % Calcium 25 % SodiumAnimal Feed

This is an example of an animal feed (broiler feed) comprising 1.5 mg/kg(1.5 ppm) of phytase variant R339D of SEQ ID NO: 2 (calculated asphytase enzyme protein):

62.55% Maize

33.8% Soybean meal (50% crude protein, CP)

1.0% Soybean oil

0.2% DL-Methionine

0.22% DCP (dicalcium phosphate)

0.76% CaCO₃ (calcium carbonate)

0.32% Sand

0.15% NaCl (sodium chloride)

1% of the above Premix

The ingredients are mixed, and the feed is pelleted at the desiredtemperature, e.g., 60, 65, 75, 80, 85, 90 or even 95° C.

Example 5: Determination of Temperature Stability

Eight variants of SEQ ID NO: 2 (the alterations as compared to SEQ IDNO: 2 are shown in Table 3 below) were prepared as described inExample 1. The two reference phytases having SEQ ID NO: 2 and SEQ ID NO:3 were prepared in the same manner.

The temperature stability was determined as follows:

200 microliter supernatants of each of the variants and the referenceproteins were split in two portions, one portion was incubated at 50° C.in plastic containers, the other was stored at 5° C. After 30 minutesincubation at 50° C. the protein solutions were transferred to anice-bath. After dilution 1:100 in 0.1 M Na-acetate buffer, pH 5.5 theactivity of the cooled and heated sample was measured by the phosphataseassay of Example 1 (“Determination of phosphatase activity”), bufferblind subtracted. The results are shown in Table 3 below as enzymeactivity (in absorption units (AU)) after incubation for 30 minutes at5° C. and 50° C., respectively, and the residual activity (RA) iscalculated as activity of the heat-treated sample (50° C. incubation)divided by the activity of the cooled sample (5° C. incubation), in %.

TABLE 3 Phytase variants with improved thermostability Phytase 5° C.(AU) 50° C. (AU) RA (%) SEQ ID NO: 2 0.210 0.070 33 SEQ ID NO: 3 1.0520.027 3 N4P of SEQ ID NO: 2 0.513 0.313 61 G5P of SEQ ID NO: 2 0.5760.287 50 Q111P of SEQ ID NO: 2 1.053 0.577 55 E1* of SEQ ID NO: 2 0.9090.401 44 E1*/E2* of SEQ ID NO: 2 0.322 0.159 50 E1*/E2*/Q3* of SEQ IDNO: 2 0.101 0.051 51 M273L of SEQ ID NO: 2 1.599 0.897 56 N286Q of SEQID NO: 2 0.062 0.024 39

Example 6: Performance in Animal Feed in an In Vitro Model

The performance in animal feed of a phytase variant is compared, in anin vitro model, to the performance of a reference protein such as SEQ IDNO: 2, SEQ ID NO: 3, SEQ ID NO: 4, and/or SEQ ID NO: 6. The in vitromodel simulates gastro-intestinal conditions in a monogastric animal andcorrelates well with results obtained in animal trials in vivo. Thecomparison is performed as follows:

Phytase activity in the variant sample is determined as described inExample 1 under “Determination of phytase activity”.

Feed samples composed of 30% soybean meal and 70% maize meal with addedCaCl₂ to a concentration of 5 g calcium per kg feed are then preparedand pre-incubated at 40° C. and pH 3.0 for 30 minutes followed byaddition of pepsin (3000 U/g feed) and suitable dosages of the phytases(identical dosages are used for all phytases to be tested to allowcomparison), for example between 0.25 to 0.75 phytase units FYT/g feed.A blank with no phytase activity is also included as reference. Thesamples are then incubated at 40° C. and pH 3.0 for 60 minutes followedby pH 4.0 for 30 minutes.

The reactions are stopped and phytic acid and inositol-phosphatesextracted by addition of HCl to a final concentration of 0.5 M andincubation at 40° C. for 2 hours, followed by one freeze-thaw cycle and1 hour incubation at 40° C.

Phytic acid and inositol-phosphates are separated by high performanceion chromatography as described by Chen et al., 203, Journal ofChromatography A 1018: 41-52 and quantified as described by Skoglund etal., 1997, J. Agric. Food Chem. 45: 431-436.

Released phosphorous is then calculated as the difference ininositol-phosphate bound phosphorous (IP-P) between phytase-treated andnon-treated samples. The relative performance of the variant iscalculated as the percentage of the phosphorous released by thereference phytase.

The in vitro performance of a number of phytase variants of SEQ ID NO: 2was determined as described above, in a dosage of 125 FYT/kg feed.

The results are shown in Tables 4A and 4B below, for supernatants andpurified phytases, respectively. Residual IP6-P designates the amount ofIP6-P (phytate phosphorous) left after the in vitro incubation and it isindicated in mg/g DM (Dry Matter). Degraded IP6-P is determined as thedifference between residual IP6-P of the blank and residual IP6-P of therespective sample. Finally, in the last column degraded IP6-P isindicated relative to the phytase having SEQ ID NO: 2. In Table 4A theblank and the reference (SEQ ID NO: 2) values are averages from a numberof independent determinations, whereas the other values are based onsingle determinations. In Table 4B the blank value is average from anumber of independent determinations, whereas the other values are basedon single determinations.

TABLE 4A In vitro performance of phytase variant supernatants VariantNo./ (amendment Residual Degraded Degraded as compared to IP6-P IP6-PIP6-P SEQ ID NO: 2) mg/g DM mg/g DM (%) Blank 2.462 026 (SEQ ID NO: 3)0.106 2.356 99 000 (SEQ ID NO: 2) 0.071 2.391 100 008 (G520/A99C) 0.3872.075 87 009 (G590/F100C) 0.272 2.190 92 010 (K141C/V199C) 0.207 2.25594 015 (N4P) 0.064 2.387 100 016 (G5P) 0.099 2.351 98 018 (Q111P) 0.1002.350 98 020 (T137P) 0.370 2.092 87 021 (L154P) 0.382 2.080 87 022(S161P) 0.235 2.227 93 023 (K240P) 0.581 1.881 79 024 (T355P) 0.7441.718 72 028 (G52E) 0.716 1.746 73 030 (E57Y) 0.666 1.796 75 032 (A84Y)0.667 1.795 75 034 (L104A) 0.709 1.753 73 035 (A105E) 0.553 1.908 80 036(K107D) 0.767 1.695 71 037 (K107G) 0.450 2.012 84 040 (E1*) 0.069 2.381100 041 (E1*/E2*) 0.095 2.367 99 042 (E1*/E2*/Q3*) 0.084 2.366 99 043(N121T) 0.423 2.039 85 044 (M273L) 0.107 2.355 98 048 (E285G) 0.5531.909 80 050 (N286Q) 0.068 2.382 100 051 (G289P) 0.560 1.902 80 052(V294T) 0.746 1.716 72 053 (I299L) 0.848 1.614 67 056 (I362K) 0.6991.763 74 059 (K107E) 0.537 1.925 80

Variants 015, 016, 018, 040, 041, 042, 044, and 050 appear to have an invitro performance which is at least as good or better than the phytasesof SEQ ID NO: 2 and 3.

TABLE 4B In vitro performance of purified phytase variants Variant No./(amendment Residual Degraded Degraded as compared to IP6-P IP6-P IP6-PSEQ ID NO: 2) mg/g DM mg/g DM (%) Blank 2.412 026 (SEQ ID NO: 3) 0.6301.782 112 102 (SEQ ID NO: 4) 0.717 1.695 106 000 (SEQ ID NO: 2) 0.8161.596 100 101 (SEQ ID NO: 9) 0.631 1.781 112 018 (Q111P) 0.843 1.569 98030 (E57Y) 0.318 2.094 131 031 (T76G) 0.384 2.028 127 037 (K107G) 0.6571.755 110 041 (E1*/E2*) 0.858 1.553 97 044 (M273L) 0.943 1.469 92 050(N286Q) 0.865 1.546 97 056 (I362K) 0.425 1.987 125 072 (I362R) 0.4301.982 124 085 (N121D) 0.555 1.856 116 087 (E196Q) 0.547 1.865 117 089(T200K) 0.405 2.007 126 090 (D202N) 0.586 1.826 114 091 (E218Q) 1.2641.148 72 095 (D314N) 0.696 1.716 107 098 (E406A) 0.515 1.897 119 1250.753 1.658 104 (Y114T/Q115Q/K116A/ D117D/E118T/E119S/K120S/N121P/D122D/ P123P/L124L) 127 0.861 1.550 97 (Y114T/Q115Q/K116T/D117D/E118T/E119S/ K120S/N121P/D122D/ P123P/L124L)

Variants 030, 031, 037, 056, 072, 085, 087, 089, 090, 095, 098, and 125appear to perform at least as good in vitro as the phytase of SEQ ID NO:3.

Example 7: Specific Activity

The specific activity of a phytase variant is determined on highlypurified samples dialysed against 250 mM sodium acetate, pH 5.5. Thepurity is checked beforehand on an SDS poly acryl amide gel showing thepresence of only one component.

The protein concentration is determined by amino acid analysis asfollows: An aliquot of the sample is hydrolyzed in 6 N HCl, 0.1% phenolfor 16 h at 110° C. in an evacuated glass tube. The resulting aminoacids are quantified using an Applied Biosystems 420A amino acidanalysis system operated according to the manufacturer's instructions.From the amounts of the amino acids the total mass—and thus also theconcentration—of protein in the hydrolyzed aliquot can be calculated.

The phytase activity is determined in the units of FYT as described inExample 1 (“Determination of phytase activity”), and the specificactivity is calculated as the phytase activity measured in FYT units permg phytase variant enzyme protein.

The specific activity for the phytase of SEQ ID NO: 2 and variant 072(1362R of SEQ ID NO: 2) was determined as described above. The specificactivity of variant 072 was 86% of the specific activity of the phytaseof SEQ ID NO: 2. The uncertainty (standard deviation) is estimated toapproximately 10%, which is mainly due to the phytase activity assaybased on a complex substrate.

Example 8: Temperature Stability

A number of variants of SEQ ID NO: 2 were prepared as described inExample 1, and the Bacillus subtilis host strains grown in 100 ml PS1medium (100 g/L sucrose, 40 g/L Soy flakes, 10 g/L Na₂HPO₄.12H₂O, 0.1ml/L Dowfax 63N10 (Dow)) in 500 ml shake flasks for four days at 30° C.at 300 rpm.

Two reference phytases were prepared in the same manner, viz. thephytase having SEQ ID NO: 3 (corresponding to variantN31D/Q139K/L197F/N316K of SEQ ID NO: 2), and the phytase having SEQ IDNO: 4 (corresponding to variant N31D/N121T/K132T/Q139K of SEQ ID NO: 2).

Also the phytase having SEQ ID NO: 9 was included for comparison(corresponding to variant Q3P/N31D/N121T/K132T/Q139K of SEQ ID NO: 2).

The temperature stability of the variants and the reference phytases wasdetermined as follows:

The supernatants were diluted ten times by adding 20 ul (microliter)supernatant to 180 ul 0.1 M Na-acetate buffer, pH 5.5+0.005% Tween-20.The diluted enzymes were split in two portions, one portion wasincubated at 60° C. in plastic containers, and the other portion wasstored at 5° C. After 30 minutes incubation at 60° C. the proteinsolutions were transferred to an ice-bath. After dilution 1:10 in 0.1 MNa-acetate buffer, pH 5.5, and 0.005% Tween-20, the activity of thecooled and heated sample was measured by the phosphatase assay ofExample 1 (“Determination of phosphatase activity”), buffer blindsubtracted.

Table 5 is a list of variants with improved temperature stability ascompared to the reference phytases. For each variant, the table alsospecifies the alterations as compared to SEQ ID NO: 2. The enzymeactivity (in absorption units (AU)) after incubation for 30 minutes at5° C. and 60° C., respectively, was determined, and the residualactivity (RA) calculated as the activity of the heat-treated sample (60°C. incubation) divided by the activity of the cooled sample (5° C.incubation). Next, the residual activity results were normalized to theresidual activity of the phytase of SEQ ID NO: 2, having been expressedand treated in the same manner. The resulting Improvement Factor (IF) isshown in Table 5. For the phytase of SEQ ID NO: 2 the IF is 1.0, whereasthe two reference phytases of SEQ ID NO: 3 and 4 were less thermostablethan the phytase of SEQ ID NO: 2, which is apparent from the fact thatthe IF for these two phytases was only 0.1 and 0.3, respectively.

TABLE 5 Phytase variants with improved thermostability No. Mutation IF026 N31D/Q139K/L197F/N316K (SEQ ID NO: 3) 0.1 101Q3P/N31D/N121T/K132T/Q139K (amino acids 23-433 of SEQ ID NO: 9) 0.3 102N31D/N121T/K132T/Q139K (SEQ ID NO: 4) 0.3 078 V409E 0.3 019 N136P 0.3082 E411K 0.4 058 E331K/V55D 0.4 086 E167Q 0.4 110K179K/T180T/T181D/E182K/K183L/S184*/T185*/K186* 0.4 059 K107E 0.4 087E196Q 0.5 070 T276R 0.5 048 E285G 0.5 053 I299L 0.5 089 T200K 0.5 065E119R 0.6 085 N121D 0.6 036 K107D 0.6 107K179K/T180E/T181K/E182H/K183Q/S184*/T185*/K186* 0.6 095 D314N 0.7 022S161P 0.7 079 T410D 0.7 004 K141C 0.7 108K179K/T180E/T181K/E182Q/K183Q/S184*/T185*/K186* 0.7 094 E285N 0.7 068R164E 0.8 081 E411R 0.8 002 G520 0.8 020 T137P 0.8 096 D314G 0.8 120 E1K0.9 042 E1*/E2*/Q3* 0.9 043 N121T 0.9 098 E406A 0.9 063 Q82E 0.9 038Q109A 0.9 000 SEQ ID NO: 2 1.0 016 G5P 1.0 030 E57Y 1.0 074 I379R 1.0041 E1*/E2* 1.0 080 T410E 1.1 040 E1* 1.2 066 E119K 1.2 028 G52E 1.2 015N4P 1.3 056 I362K 1.3 090 D202N 1.3 071 T276K 1.3 076 N385D 1.3 113Q111P/E241Q 1.4 005 S162C 1.4 109K179K/T180E/T181K/E182K/K183V/S184*/T185*/K186* 1.4 093 E241Q 1.4 069Q223E 1.5 050 N286Q 1.5 037 K107G 1.5 125Y114T/Q115Q/K116A/D117D/E118T/E119S/K120S/N121P/D122D/P123P/L124L 1.6075 I379K 1.6 044 M273L 1.6 006 N31C 1.7 083 E53V 1.8 009 G59C/F100C 1.9062 W46E 2.2 018 Q111P 2.2 127Y114T/Q115Q/K116T/D117D/E118T/E119S/K120S/N121P/D122D/P123P/L124L 2.3031 T76G 2.3 072 I362R 2.7 010 K141C/V199C 4.3 008 G52C/A99C 5.2

Example 9: Thermostability by DSC

A number of purified variants of SEQ ID NO: 2 were prepared as generallydescribed in Example 1. Two reference phytases were prepared in the samemanner, viz. the phytase having SEQ ID NO: 3 (corresponding to variantN31D/Q139K/L197F/N316K of SEQ ID NO: 2), and the phytase having SEQ IDNO: 4 (corresponding to variant N31D/N121T/K132T/Q139K of SEQ ID NO: 2).Also the phytase having SEQ ID NO: 9 was included for comparison(corresponding to variant Q3P/N31D/N121T/K132T/Q139K of SEQ ID NO: 2).

Aliquots of the protein samples were dialysed against 2×500 ml 20 mMNa-acetate, pH 4.0 at 4° C. in a 2-3 h step followed by an over nightstep. Each sample was 0.45 um filtered and diluted with buffer toapprox. 2 A₂₈₀ units. The exact absorbance values measured are given inthe results table. DSC was performed on a MicroCal VP-DSC at 90° C./hscan rate from 20-90° C. in 20 mM Na-acetate buffer, pH 4.0.

The resulting denaturation temperatures (Td) are shown in Table 6 below,which summarizes the results of three different experiments.

TABLE 6 Td measurements by DSC Phytase A₂₈₀ T_(d) (° C.) Comments No.102 (SEQ ID NO: 4) 2.0 61.5 No. 000 (SEQ ID NO: 2) 2.4 61.2 No. 101(amino acids 23-433 of SEQ 2.3 61.0 ID NO: 9) No. 031 (T76/G of SEQ IDNO: 2) 2.0 61.3 No. 056 (I362K of SEQ ID NO: 2) 2.4 62.1 No. 072 (I362Rof SEQ ID NO: 2) 2.4 61.6 Minor precipitate No. 000 (SEQ ID NO: 2) 2.2961 No. 018 (Q111P of SEQ ID NO: 2) 1.97 62 YC044 (M273L of SEQ ID NO: 2)2.15 62 No. 000 (SEQ ID NO: 2, pilot 1.35 62 fermentation) No. 026 (SEQID NO: 3) 1.99 57 Run twice with same result

Example 10: Purification and Temperature Profile

The phytase variants and reference and comparative phytases used hereinwere purified as follows: The fermentation supernatant with the phytasewas first centrifuged at 7200 rpm and 5° C. for one hour and filteredthrough a sandwich of four Whatman glass microfibre filters (2.7, 1.6,1.2 and 0.7 micrometer). Following this the solution was sterilefiltered (either through a Fast PES Bottle top filter with a 0.22 μmcut-off or through a Seitz-EKS depth filter using pressure). Thesolution was added solid ammonium sulfate giving a final concentrationof 1.5 M and the pH was adjusted to 6.0 using 6 M HCl.

The phytase-containing solution was applied to a butyl-sepharose column,approximately 50 ml in a XK26 column, using as buffer A 25 mM bis-tris(Bis-(2-hydroxyethyl)imino-tris(hydroxymethyl)methan))+1.5 M ammoniumsulfate pH 6.0, and as buffer B 25 mM bis-tris pH6.0. The fractions fromthe column were analyzed for activity using the phosphatase assay (seeExample 1, “Determination of phosphatase activity”) and fractions withactivity were pooled. The pooled fractions were dialyzed extensivelyagainst 10 mM sodium acetate pH 4.5. Following this thephytase-containing solution was purified by chromatography on SSepharose, approximately 75 ml in a XK26 column, using as buffer A 50 mMsodium acetate pH 4.5, and as buffer B 50 mM sodium acetate+1 M NaCl pH4.5. Again the fractions from the column were analyzed for activity andfractions with activity were pooled. Finally, the solution containingthe purified phytase was concentrated using an Amicon ultra-15 filteringdevice with a 10 kDa cut-off membrane.

The molecular weight, as estimated from SDS-PAGE, was approximately 40kDa for all phytases and the purity was in all cases>95%.

The temperature profile (phytase activity as a function of temperature)of the variants was determined in the temperature range of 20-90° C.essentially as described in Example 1 (“Determination of phytaseactivity”), however, the enzymatic reactions (100 microlitersphytase-containing enzyme solution+100 microliters substrate) wereperformed in PCR tubes instead of microtiter plates. After a 15 minutereaction period at desired temperature the tubes were cooled to 20° C.for 20 seconds and 150 microliter of the reaction mixture wastransferred to a microtiter plate. 75 microliter stop reagent was addedand the absorbance at 405 nm was measured in a microtiter platespectrophotometer. The results are summarized in Table 7 below. Thenumbers given for each temperature (20-90° C. in 10° C. steps) arerelative activity (in %) normalized to the value at optimum.

TABLE 7 Temperature profiles Temperature (° C.) No. Phytase 20 30 40 5060 70 80 90 026 SEQ ID NO: 3 18 32 52 74 100 4 4 −1 102 SEQ ID NO: 4 2237 55 79 100 17 10 4 000 SEQ ID NO: 2 20 34 54 68 100 21 11 5 101 Aminoacids 23-433 of 16 28 50 65 100 12 6 −1 SEQ ID NO: 9 018 Q111P of SEQ IDNO: 2 20 34 53 75 100 17 10 4 030 E57Y of SEQ ID NO: 2 21 35 57 79 10028 10 4 031 T76G of SEQ ID NO: 2 20 34 55 77 100 23 10 4 037 K107G ofSEQ ID NO: 2 21 34 55 77 100 32 11 4 041 E1*/E2* of SEQ ID NO: 2 21 3456 78 100 15 9 4 044 M273L of SEQ ID NO: 2 21 35 56 79 100 26 9 3 050N286Q of SEQ ID NO: 2 16 32 44 84 100 6 −1 −7 056 I362K of SEQ ID NO: 220 35 54 78 100 25 9 3 062 W46E of SEQ ID NO: 2 27 42 65 84 100 18 12 5072 I362R of SEQ ID NO: 2 21 35 56 79 100 23 11 5 083 E53V of SEQ ID NO:2 20 32 53 76 100 22 10 5 093 E241Q of SEQ ID NO: 2 22 37 59 83 100 2010 3

Variants 030, 031, 037, 044, 056, 062, 072, 083, and 093 have a higherrelative activity at 70° C. as compared to the reference phytases 026and 102.

Example 11: pH Profile

The pH profiles (phytase activity as a function of pH) of a number ofvariants and the same reference and comparative phytases as used in theprevious examples were determined at 37° C. in the pH range of 2.0 to7.5 (in 0.5 pH-unit steps) as described in Example 1 (“Determination ofphytase activity”), except that a buffer cocktail (50 mM glycine, 50 mMacetic acid and 50 mM Bis-Tris was used instead of the 0.25 M sodiumacetate pH 5.5 buffer. The results are summarized in Table 8 below. Thenumbers given for each pH (2.0-7.5) are relative activity (in %)normalized to the value at optimum.

TABLE 8 pH profiles pH No. Phytase 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.06.5 7.0 7.5 026 SEQ ID NO: 3 30 62 87 99 100 95 84 65 40 14 −1 −2 102SEQ ID NO: 4 26 65 87 97 100 93 82 66 43 17 0 −4 000 SEQ ID NO: 2 33 6089 100 99 95 80 61 35 11 −2 −4 101 Amino acids 23-433 of SEQ ID NO: 9 3765 87 97 100 93 80 65 42 19 2 0 018 Q111P of SEQ ID NO: 2 27 61 89 97100 90 75 57 32 10 −2 −4 030 E57Y of SEQ ID NO: 2 35 60 86 97 100 92 7960 36 13 1 −3 031 T76G of SEQ ID NO: 2 34 60 86 99 100 93 79 62 36 12 1−1 037 K107G of SEQ ID NO: 2 33 59 87 100 99 93 78 60 34 10 −2 −4 041E1*/E2* of SEQ ID NO: 2 29 62 89 100 100 93 79 59 34 12 2 1 044 M273L ofSEQ ID NO: 2 31 63 87 100 99 94 81 62 36 12 0 −1 050 N286Q of SEQ ID NO:2 29 59 87 100 97 89 77 56 36 12 1 −2 056 I362K of SEQ ID NO: 2 34 60 85100 98 92 74 64 35 12 4 1 062 W46E of SEQ ID NO: 2 13 40 77 100 95 88 7252 29 10 1 −1 072 I362R of SEQ ID NO: 2 35 60 87 100 100 97 82 64 37 141 −1 083 E53V of SEQ ID NO: 2 22 61 88 99 100 94 80 61 35 9 −6 −5 085N121D of SEQ ID NO: 2 34 65 91 100 94 82 65 46 27 11 4 0 087 E196Q ofSEQ ID NO: 2 30 63 89 100 99 89 73 54 28 8 4 0 089 T200K of SEQ ID NO: 225 58 86 100 92 78 61 43 18 4 0 −1 090 D202N of SEQ ID NO: 2 30 66 91100 99 91 70 46 18 3 −1 −3 091 E218Q of SEQ ID NO: 2 11 45 75 95 100 9590 64 35 8 −2 −4 093 E241Q of SEQ ID NO: 2 28 58 83 92 100 87 71 59 31 8−1 −3 095 D314N of SEQ ID NO: 2 26 60 87 100 98 94 75 58 33 11 2 0 098E406A of SEQ ID NO: 2 32 59 89 100 98 91 76 59 30 9 2 −3 125Y114T/Q115Q/K116A/D117D/E118T/E119S/K120S/N121P/D122D/P123P/L124L 30 6687 98 100 86 70 54 29 10 1 −1 of SEQ ID NO: 2 127Y114T/Q115Q/K116T/D117D/E118T/E119S/K120S/N121P/D122D/P123P/L124L 42 6791 100 100 90 74 55 27 8 −2 −4 of SEQ ID NO: 2

For YC062 and YC091 the pH curve (relative activity as a function of pH)seems to have shifted 0.5 pH unit towards higher pH.

Furthermore, while for most of the Table 8 phytases (including thereference phytases 026 and 102) the optimum is at pH3.5-pH4.0, anoptimum pH of 3.5 is observed for no. 062, 085, and 089, and an optimumpH of 4.0 is observed for no. 091 and 093.

Example 12: Temperature Stability

The temperature stability of a number of purified variants and the samereference and comparative phytases as in the previous examples wasdetermined by measuring residual phytase activity after incubation at70° C. and pH 4.0 (0.1 M sodium acetate). The phytases were incubatedand samples were withdrawn after 0, 10, 30 and 60 minutes and cooled onice. The residual activity at pH 5.5 was determined using the methoddescribed in Example 1 (“Determination of phytase activity”). Theresults, normalized to the activity found at 0 minutes, are shown inTable 9 below.

TABLE 9 Temperature stability Temperature stability No. Phytase % after60 min. 026 SEQ ID NO: 3 26 102 SEQ ID NO: 4 30 000 SEQ ID NO: 2 46 101Amino acids 23-433 of 29 SEQ ID NO: 9 018 Q111P of SEQ ID NO: 2 25 044M273L of SEQ ID NO: 2 31 050 N286Q of SEQ ID NO: 2 19 056 I362K of SEQID NO: 2 26 062 W46E of SEQ ID NO: 2 31 072 I362R of SEQ ID NO: 2 32 083E53V of SEQ ID NO: 2 31 093 E241Q of SEQ ID NO: 2 29

The above results indicate that Nos. 044, 062, 072, and 083 may be morestable under these conditions (70° C. and pH 4) than the referencephytases (although in this experiment a big variation was observed forNo. 000).

Example 13: Calculating Percentage of Identity and IdentifyingCorresponding Positions

SEQ ID NO: 9 was aligned with SEQ ID NO: 2 using the Needle program fromthe EMBOSS package version 2.8.0. The substitution matrix used wasBLOSUM62, the gap opening penalty was 10.0, and the gap extensionpenalty was 0.5.

The resulting alignment is shown in FIG. 2.

The degree of identity between SEQ ID NO: 9 and SEQ ID NO: 2 iscalculated as follows: The number of exact matches is 406 (all thosewith a vertical stroke). The length of the shortest sequence is 411 (SEQID NO: 2). The percentage of identity is 406/411×100%=98.8%.

The alignment of FIG. 2 is also used for deriving correspondingpositions as follows: Amino acids on top of each other in this alignmentare in corresponding positions. For example, amino acid Q in position 3of SEQ ID NO: 2 corresponds to amino acid P in position number 25 of SEQID NO: 9. For the present purposes we refer to the position number ofSEQ ID NO: 2. Therefore, SEQ ID NO: 9 may be considered a variant of SEQID NO: 2 which comprises the substitution Q3P.

Other differences in the form of substitutions within the overlap of thealignment are found in positions 31, 121, 132, and 139, viz. N31D,N121T, K132T, and Q139K.

Additional differences are found in the N-terminus, where SEQ ID NO: 9has an extension of 22 amino acids as compared to SEQ ID NO: 2.

Overall, SEQ ID NO: 9 may therefore be considered the following variantof SEQ ID NO: 2:*0aM/*0bS/*0cT/*0dF/*0el/*0fl/*0gR/*0hL/*0iL/*0jF/*0kF/*0mS/*0nL/*0oL/*0pC/*0qG/*0rS/*0sF/*0tS/*0ul/*0vH/*0wA/Q3P/N31D/N121T/K132T/Q139K.

The invention claimed is:
 1. A phytase variant comprising at least onesubstitution as compared to SEQ ID NO: 2 in at least one positionselected from the group consisting of: 3, 5, 41, 82, 84, 105, 109, 136,137, 161, 164, 167, 171, 179, 180, 186, 218, 239, 240, 281, 282, 283,284, 289, 294, 299, 308, 314, 324, 331, 339, 351, 355, 409, 410, and411; and which has at least 80% identity to SEQ ID NO: 2 and providedthat the phytase variant is not SEQ ID NO: 3, not SEQ ID NO: 4, and notSEQ ID NO:
 6. 2. The phytase variant of claim 1, further comprising asubstitution at position
 203. 3. The phytase variant of claim 1, whereinthe at least one substitution comprises a substitution selected from thegroup consisting of 5P, 41P, 82E, 84Y, 105F, 109A,G, 137P, 161P, 164D,E,167Q, 171T, 179G,I,K,N,Q, 180A,E,G,T, 218Q, 239Q, 240P, 281H, 282P,283P, 284P, 294T, 299L, 308A, 314G,N, 324N, 331K, 339D, 351Y, 355P,409D,E, 410D, E, and/or 411R, K.
 4. The phytase variant of claim 1,further comprising a substitution in at least one position selected fromthe group consisting of 1, 2, 4, 31, 46, 52, 53, 55, 57, 59, 74, 76, 91,99, 100, 104, 107, 111, 114, 115, 116, 117, 118, 119, 120, 121, 122,123, 124, 141, 154, 162, 176, 177, 181, 182, 183, 184, 185, 196, 199,200, 202, 223, 241, 247, 273, 276, 285, 286, 316, 362, 379, 385, and406.
 5. The phytase variant of claim 1, which has at least 85% identityto SEQ ID NO:
 2. 6. The phytase variant of claim 1, which has at least90% identity to SEQ ID NO:
 2. 7. The phytase variant of claim 1, whichhas at least 95% identity to SEQ ID NO:
 2. 8. A composition comprisingthe phytase variant of claim 1, and at least one of (a) at least one fatsoluble vitamin; (b) at least one water soluble vitamin; and (c) atleast one trace mineral.
 9. The composition of claim 8, furthercomprising at least one enzyme selected from the group consisting ofamylase, galactanase, alpha-galactosidase, beta-glucanase, phosphatase,phospholipase, phytase, protease, and/or xylanase.
 10. A compositioncomprising the phytase variant of claim 5, and at least one of (a) atleast one fat soluble vitamin; (b) at least one water soluble vitamin;and (c) at least one trace mineral.
 11. A composition comprising thephytase variant of claim 6, and at least one of (a) at least one fatsoluble vitamin; (b) at least one water soluble vitamin; and (c) atleast one trace mineral.
 12. A composition comprising the phytasevariant of claim 7, and at least one of (a) at least one fat solublevitamin; (b) at least one water soluble vitamin; and (c) at least onetrace mineral.
 13. A phytase variant comprising at least one set ofsubstitutions selected from the group consisting of: Q3P/N31 D/N121T/K132T/Q139K, N31 D/Q139K/L197F/N316K, 55D/331 K, 104A/105F,280P/282P/283P, and the substitution of amino acids K179, T180, T181,E182, K183, S184, T185, and K186 with QADKP (SEQ ID NO: 17), GEDKP (SEQID NO: 18), NGISA (SEQ ID NO: 19), IAGKS (SEQ ID NO: 20), KEKHQ (SEQ IDNO: 21), KEKQQ (SEQ ID NO: 22), KEKKV (SEQ ID NO: 23), or KTDKL (SEQ IDNO: 24).
 14. An animal feed composition having a crude protein contentof 50 to 800 g/kg and comprising the phytase variant of claim
 1. 15. Amethod for improving the nutritional value of an animal feed, whereinthe phytase variant of claim 1 is added to the feed.
 16. A process forreducing phytate levels in animal manure comprising feeding an animalwith an effective amount of the animal feed composition of claim 14.